Methods and compositions for cell and tissue rejuvenation

ABSTRACT

The present disclosure provides compositions, methods and kits for the rejuvenation of target cells. In some aspects, the compositions, methods and kits comprise mRNAs the promote the expression of TERT and/or TERC.

RELATED APPLICATIONS

This application claims priority to, and the benefit of, U.S. Provisional Application No. 62/899,861, filed Sep. 13, 2019, the contents of which is incorporated herein by reference in their entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Aug. 31, 2020, is named “UNCO-028_001WO_SeqList.txt” and is about 241 KB in size.

BACKGROUND OF THE INVENTION

The shortening of telomeres, repetitive DNA sequences at the ends of linear chromosomes, can lead to cellular senescence, apoptosis, or malignancy. In particular, the shortening of telomeres in cells cultured in vitro is an obstacle to the production of therapeutic cell populations, as shortened telomeres can limit further expansions of the therapeutic cell populations as well as degrade the cells' biological activity, leading to a decrease clinical efficacy. Increasing telomere length in cells can lead to cellular rejuvenation, but can also cause deleterious side-effects such as oncogenic cellular immortalization. Thus, there is a need in the art for compositions, kits and methods directed to effectively and safely increasing the length of telomeres in cells in a controllable way, thereby rejuvenating the cells.

SUMMARY OF THE INVENTION

The present disclosure provides a composition comprising: a) at least one first polynucleotide molecule comprising a nucleic acid sequence encoding at least a portion of telomerase reverse transcriptase (TERT); and b) at least one second polynucleotide molecule comprising a nucleic acid sequence encoding at least a portion of at least one DNA targeting polypeptide, wherein the DNA targeting polypeptide increases transcription of telomerase RNA component (TERC).

The present disclosure provides a composition comprising: a) at least one first polynucleotide molecule comprising a nucleic acid sequence encoding at least a portion of telomerase reverse transcriptase (TERT); and b) at least one DNA targeting polypeptide, wherein the DNA targeting polypeptide increases transcription of telomerase RNA component (TERC).

A first polynucleotide molecule can comprise an mRNA molecule encoding at least a portion of TERT. A first polynucleotide molecule can comprise a plasmid comprising a nucleic acid sequence encoding at least a portion of TERT operably linked to at least one promoter sufficient to drive expression of the at least one portion of TERT.

A second polynucleotide molecule can comprise an mRNA molecule encoding at least a portion of at least one DNA targeting polypeptide. A second polynucleotide molecule can comprise a plasmid comprising a nucleic acid sequence encoding at least a portion of at least one DNA targeting polypeptide operably linked to at least one promoter sufficient to drive expression of the at least one portion of the at least one DNA targeting polypeptide.

A DNA targeting polypeptide can comprise at least one Cas9 molecule, at least one Cas9 variant molecule, at least one Cas9 ortholog molecule or any combination thereof. A Cas9 molecule, a Cas9 variant molecule or a Cas9 ortholog molecule can be nuclease-deficient or nuclease-dead. A Cas9 variant molecule can comprise eSpCas9 (K855A), eSpCas9 (1.0), eSpCas9 (1.1), SpCas9-HF1 (VP12), HypaCas9, xCas9, SpyFi Cas9, iSpy Cas9, iSpyMac, Cas9 (VQR), Cas9 (EQR), Cas9 (VRER), Cas9 (D1135E), Cas9(QQR1), SaCas9 (KKH), Nmel Cas9, Nme2Cas9, Nme3Cas9 or any combination thereof. A Cas9 ortholog molecule can comprise Streptococcus pyogenes Cas9 (spCas9), Francisella novicida Cas9 (FnCas9), Staphylococcus aureus Cas9 (SaCas9), Neisseria meningitidis Cas9 (NmCas9; NmeCas9), Streptococcus thermophilus CRISPR1-Cas9 (St1Cas9), Streptococcus thermophilus CRISPR3-Cas9 (St3Cas9), Campylobacter jejuni Cas9 (CjCas9), Acidaminococcus sp. BV3L6 Cpf1 (AsCpf1), Lachnospiraceae bacterium ND2006 Cpfl (LbCpfl), Streptococcus canis Cas9 (ScCas9), Treponema denticola Cas9 (TdCas9), Streptococcus macacae Cas9 (SmacCas9), Casϕ (Cas12j), Francisella tularensis subsp. novicida Cas9, Pasteurella multocida Cas9, Campylobacter lari CF89-12 Cas9, Mycoplasma gallisepticum str. F Cas9, Nitratifractor salsuginis str DSM 16511 Cas9, Parvibaculum lavamentivorans Cas9, Roseburia intestinalis Cas9, Neisseria cinerea Cas9, Gluconacetobacter diazotrophicus Cas9, Azospirillum B510 Cas9, Sphaerochaeta globus str. Buddy Cas9, Flavobacterium columnare Cas9, Fluviicola taffensis Cas9, Bacteroides coprophilus Cas9, Mycoplasma mobile Cas9, Lactobacillus farciminis Cas9, Streptococcus pasteurianus Cas9, Lactobacillus johnsonii Cas9, Staphylococcus pseudintermedius Cas9, Filifactor alocis Cas9, Legionella pneumophila str. Paris Cas9, Sutterella wadsworthensis Cas9, Corynebacter diphtheriae Cas9 or any combination thereof.

A DNA targeting polypeptide can comprise at least one TALE molecule, at least one zinc-finger molecule, at least one meganuclease molecule or any combination thereof

A DNA targeting polypeptide can comprise at least one transactivation molecule. A transactivation molecule can comprise at least one P65 molecule, at least one Rta molecule, at least one VP16 molecule, at least one VP64 molecule, at least one VP160 molecule, at least one VP64-P65-Rta (VPR) molecule, at least one SunTag peptide, at least one single guide RNA-MS2 (sgRNA-MS2) molecule or any combination thereof. In some aspects, a DNA targeting polypeptide can be a DNA targeting ribonucleoprotein (RNP) complex. A DNA targeting ribonucleoprotein complex can comprise both at least one protein component and at least one nucleic acid component. A DNA targeting polypeptide can comprise at least one guide RNA. A transactivation molecule can comprise at least one single guide RNA-MS2 (sgRNA-MS2) molecule. An sgRNA-MS2 molecule can comprise a nucleic acid sequence complementary to a nucleic acid sequence located upstream, within, or downstream of the endogenous TERC gene and at least about one, or at least about two, or at least about three, or at least about four, or at least about five, or at least about six, or at least about seven, or at least about eight, or at least about nine, or at least about ten MS2 RNA aptamers.

A DNA targeting polypeptide can comprise a dCas9 molecule and a VPR molecule.

A DNA targeting polypeptide can bind upstream of, 5′ to, within, downstream of or 3′ to the endogenous TERC gene.

An mRNA molecule can be a modified mRNA molecule. A modified mRNA molecule can comprise at least one modified ribonucleoside base. A modified ribonucleoside base can comprise a pseudouridine (Ψ) residue, a 5-methylcytidine (m⁵C) residue or any combination thereof. A modified mRNA molecule can comprise at least one modified nucleoside. A modified nucleoside can comprise 5-methylcytidine (m⁵C), 5-methyluridine (m⁵U), N6-methyladenosine (m⁶A), inosine 2′-0-methylated nucleosides or any combination thereof.

Any composition of the present disclosure can further comprise a plurality of guide RNA (gRNA) molecules, wherein at least one gRNA in the plurality is complementary to a nucleic acid sequence located upstream, within, or downstream of the endogenous TERC gene. A plurality of gRNA molecules can comprise at least about one, or at least about two, or at least about three, or at least about four, or at least about five, or at least about six, or at least about seven, or at least about eight, or at least about nine, or at least about ten distinct species of gRNA molecules, wherein each species has a different nucleic acid. Any composition of the present disclosure can further comprise at least one plasmid comprising at least one nucleic acid sequence encoding at least one species of gRNA operably linked to at least one promoter sufficient to drive expression of the at least one species gRNA. A plurality of gRNA molecules can comprise a plurality of single guide RNA (sgRNA) molecules, crRNA:tracrRNA molecules, truncated sgRNA molecules, high fidelity scaffold gRNA molecules or any combination thereof. A guide RNA molecule can be a modified guide RNA (mod gRNA) molecule. A guide RNA molecule can comprise any sequence recited in Table 1 or Table 2.

The present disclosure provides a composition comprising: a) at least one modified mRNA molecule comprising a nucleic acid sequence encoding at least a portion of human telomerase reverse transcriptase (hTERT); b) at least one modified mRNA molecule comprising a nucleic acid sequence encoding at least a portion of at least one DNA targeting polypeptide, wherein the at least one DNA targeting polypeptide comprises dCas9 and a VP64-P65-Rta (VPR) molecule; and c) a plurality of guide RNA (gRNA) molecules, wherein at least one gRNA in the plurality is complementary to a nucleic acid sequence located upstream of the endogenous hTERC gene.

Any composition of the present disclosure can comprise at least one mRNA and/or polynucleotide encoding at least one rejuvenating factor. A rejuvenating factor can comprise telomerase RNA component (TERC), telomerase associated reverse-transcriptase (TERT), protection of telomeres 1 (POT1), insulin-like growth factor 1 (IGF1), WD repeat containing antisense to TP53 (WRAP53), nuclear protein family A, member 3 (NOP3), heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1), shelterin complex subunit and telomerase recruitment factor (ACD/TPP1), TRF-1 interacting ankyrin-related ADP-ribose polymerase (TNKS), telomeric repeat binding factor 1 (TRF-1), telomeric repeat binding factor 2 (TRF-2), TERF1 interacting nuclear factor 2 (TIN2), telomeric repeat binding factor 2 (Rap1), Dyskerin Pseudouridine Synthase 1 (DKC1), ribonucleoprotein NHP2 or any combination thereof.

TERT can be human TERT (hTERT). TERC can be human T ERC (hTERC).

The present disclosure provides a composition comprising at least one viral particle comprising any composition of the present disclosure. A viral particle can be an adeno-associated virus (AAV) particle, adenovirus particle, lentivirus particle, foamy-virus particle, herpes simplex virus (HSV) particle, retrovirus particle, alphavirus particle, flavivirus particle, rhabdovirus particle, measle virus particle, Newcastle disease virus particle, poxvirus particle, picornavirus particle, or any combination thereof. An AAV particle can be an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV2/1, AAV2/2, AAV2/3, AAV2/4, AAV2/5, AAV2/6, AAV2/7, AAV2/8, AAV2/9, AAV-DJ or AAV-DJ8 particle. A retrovirus particle can be MMSV or MSCV particle. A lentivirus particle can be HIV-1 or HIV-2 particle. An alphavirus particle can be SFV, SIN, VEE, or M1 particle. A flavivirus particle can be Kunjin virus, West Nile virus, or Dengue virus particle.

The present disclosure provides a composition comprising at least one exosome, microvesicle or liposome, wherein the at least one exosome, microvesicle or liposome comprises any composition of the present disclosure. The present disclosure provides a composition comprising least one nanoparticle, wherein the at least one nanoparticle comprises any composition of the present disclosure. A nanoparticle can comprise a liposome, a micelle, a polymer-based nanoparticle, a lipid-polymer based nanoparticle, a metal based nanoparticle, a nanocrystal, a carbon nanotube based nanoparticle or a polymeric micelle.

The present disclosure provides a kit comprising any composition of the present disclosure.

The present disclosure provides a method of rejuvenating at least one cell, the method comprising contacting the at least one cell with any composition or kit of the present disclosure. The preceding method can further comprise expanding the at least one cell contacted with any composition or kit of the present disclosure to produce a plurality of rejuvenated cells.

The present disclosure provides a method of treating and/or preventing a disease in a subject comprising: a) contacting at least one cell with any composition or kit of the present disclosure; b) expanding the at least one cell contacted with any composition or kit of the present disclosure to produce a plurality of rejuvenated cells; and c) administering the plurality of rejuvenated cells to the subject.

The present disclosure provides a method of treating and/or preventing a disease in a subject comprising: a) contacting at least one cell with any composition or kit of the present disclosure ; b) expanding the at least one cell contacted with any composition or kit of the present disclosure to produce a plurality of rejuvenated cells; c) culturing the plurality of rejuvenated cells under conditions sufficient to transform the plurality of rejuvenated cells into at least one tissue or organ; and d) administering the at least one tissue or organ to the subject.

The present disclosure provides a method of producing an in vitro tissue or organ comprising: a) contacting at least one cell with any composition or kit of the present disclosure; b) expanding the at least one cell contacted with any composition or kit of the present disclosure to produce a plurality of rejuvenated cells; c) culturing the plurality of rejuvenated cells under conditions sufficient to transform the plurality of rejuvenated cells into at least one tissue or organ.

The present disclosure provides a method of producing a plurality of rejuvenated edited cells comprising: a) contacting a plurality of cells with a gene editing system such that at least one gene in the genome of at least one cell in the plurality is edited, thereby producing at least one edited cell; b) isolating the at least one edited cell; c) contacting the isolated at least one edited cell with any composition or kit of the present disclosure; and d) expanding the at least one cell contacted with any composition or kit of the present disclosure to produce a plurality of rejuvenated edited cell.

The present disclosure provides a method of treating and/or preventing a disease in a subject comprising: a) contacting a plurality of cells with a gene editing system such that at least one gene in the genome of at least one cell in the plurality is edited, thereby producing at least one edited cell; b) isolating the at least one edited cell; c) contacting the isolated at least one edited cell with any composition or kit of the present disclosure; d) expanding the at least one cell contacted with any composition or kit of the present disclosure to produce a plurality of rejuvenated edited cells; and e) administering to the subject the plurality of rejuvenated edited cells.

The present disclosure provides a method of treating epidermolysis bullosa (EB) in a subject comprising: a) contacting a plurality of cells comprising keratinocytes, dermal fibroblasts, mesenchymal stem/stromal cells or any combination thereof with a gene editing system such that at least one gene in the genome of at least one cell in the plurality is edited, thereby producing at least one edited cell; b) isolating the at least one edited cell; c) contacting the isolated at least one edited cell with any composition or kit of the present disclosure; d) expanding the at least one cell contacted with any composition or kit of the present disclosure to produce a plurality of rejuvenated edited cells; and e) administering to the subject the plurality of rejuvenated edited cells.

Expanding the at least one cell can comprise culturing the at least one cell using adjusted Opti-MEM, non-adjusted Opti-MEM, human serum, fetal bovine serum (FBS) or any combination thereof.

Rejuvenating at least one cell comprises can increase the expression of TERC in the at least one cell, increasing the expression of TERT in the at least one cell, increasing the total number of population doublings exhibited by the at least one cell, increasing the length of telomeres in the at least one cell, increasing the mitochondrial DNA copy number in the at least one cell, increasing the amount of mitochondrial DNA in the at least one cell, increasing the number of mitochondria in the at least one cell, increasing the migration activity of the at least one cell, restoring the young-like state of thiol group oxidation levels in proteins in the at least one cell, reducing senescence-associated DNA methylation in the at least one cell or any combination thereof.

An at least one cell can be a fibroblast, a keratinocyte, a mesenchymal stem/stromal cell, a peripheral blood mononuclear cell, a chimeric antigen receptor T cell (CAR-T cell), an endothelial cell, a chondrocyte, a muscle stem cell, a neural stem cell, a hepatocyte, a limbal stem cell, a retinal pigmented epithelial cell, a hematopoietic stem cell, a macrophage, a cardiomyocyte, a pancreatic cell, a β-cell or any combination thereof.

A disease can comprise graft-vs-host diseases (GvHD), autoimmune diseases, epidermolysis bullosa (EB), recessive dystrophic form of EB (RDEB), junctional EB (JEB), EB simplex (EBS), congenital ichthyosis, congenital dyskeratosis, macular degeneration, Parkinson's disease, Alzheimer's disease, aging, Type I and II diabetes, burns, chronic skin wounds, diabetes-associated ulcers/wounds, heart disease, osteoporosis, cancer, connective tissue diseases such as Ehlers-Danlos Syndrome (EDS) or Marfan syndrome, liver diseases, lung diseases, and any combination thereof.

Contacting at least one cell can comprise transfection, transduction, electroporation, nucleofection, at least one cell-penetrating peptide or any combination thereof.

The present disclosure provides a method for rejuvenating at least one cell in a subject comprising administering to the subject at least one therapeutically effective amount of any composition or kit of the present disclosure.

The present disclosure provides a method for rejuvenating at least one subject comprising administering to the subject at least one therapeutically effective amount of any composition or kit of the present disclosure.

A subject can be a mammal. A subject can be a human, a primate, a mouse, a rat, a dog, a cat, a cow, a horse, a goat, a camel, a sheep, a pig or any other mammal. A subject can be a bird.

Any of the above aspects can be combined with any other aspect.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. In the Specification, the singular forms also include the plural unless the context clearly dictates otherwise; as examples, the terms “a,” “an,” and “the” are understood to be singular or plural and the term “or” is understood to be inclusive. By way of example, “an element” means one or more element. Throughout the specification the word “comprising,” or variations such as “comprises” or “comprising,” will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps. About can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from the context, all numerical values provided herein are modified by the term “about.”

Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. The references cited herein are not admitted to be prior art to the claimed invention. In the case of conflict, the present Specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be limiting. Other features and advantages of the disclosure will be apparent from the following detailed description and claim.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and further features will be more clearly appreciated from the following detailed description when taken in conjunction with the accompanying drawings.

FIG. 1 is a schematic of a DNA-targeting molecule of the present disclosure binding upstream of an endogenous hTERC locus. In this non-limiting example, the DNA-targeting molecule comprises dCas9 and a transactivation molecule, wherein the transactivation molecule is a VP64-P65-Rta (VPR) molecule.

FIG. 2 is a schematic overview of a treatment method and/or a method of producing an in vitro tissue of the present disclosure

FIG. 3 is a schematic overview of a method of producing a plurality of rejuvenated edited cells of the present disclosure.

FIG. 4 is a chart showing the hTERC transcript level in various cell types.

FIG. 5 is a chart showing the level of human TERC RNA in F50 cells transfected with compositions of the present disclosure.

FIG. 6 is a series of charts showing the level of human TERC RNA in HEKn cells (left) and human Mesenchymal Stem/Stromal Cells (hMSCs) (right) transfected with compositions of the present disclosure (+dCas9−VPR+gmix) as compared to non-transfected HEKn cells, non-transfected hMSC cells and F50-derived induced pluripotent stem cells.

FIG. 7 is a series of charts showing the level of human TERC RNA in F50 cells (left) and hMSCs (right) transfected with compositions of the present disclosure lacking guide RNA (+dCas9−VPR (no guide)).

FIG. 8 is schematic overview of one of the transfection regimes of the present disclosure.

FIG. 9 is a chart showing the total population doubling of senescent F50S cells transfected with compositions of the present disclosure.

FIG. 10 is a schematic overview of an alternative transfection regime of the present disclosure.

FIG. 11 is a schematic overview of another transfection regime of the present disclosure.

FIG. 12 is a chart showing the relative telomere length in senescent F50S cells transfected with compositions of the present disclosure (+TERT+dCas9−VPR/gmix) as compared to non-transfected F50S cells and F50-derived induced pluripotent stem cells.

FIG. 13 is a chart showing the relative telomere length in F50 cells transfected with compositions of the present disclosure (+hTERT+dCas9−VPR/gmix) as compared to non-transfected F50 cells and F50-derived induced pluripotent stem cells.

FIG. 14 is a series of charts showing the relative telomere length in HEKn cells (left) and hMSCs (right) transfected with compositions of the present disclosure (+TERT+dCas9−VPR/gmix) as compared to non-transfected HEKn cells, non-transfected hMSCs and F50-derived induced pluripotent stem cells.

FIG. 15 is a series of charts showing the relative amount of mitochondrial DNA in F50 cells (left), HEKn cells (middle) and hMSCs (right) transfected with compositions of the present disclosure (+TERT+dCas9−VPR/gmix) as compared to non-transfected F50 cells, non-transfected HEKn cells and non-transfected hMSCs.

FIG. 16 is a gel image of the results of a telomerase activity assay in F50 cells transfected with various compositions of the present disclosure (F50+TERT and F50+TERT+dCas9−VPR/gmix) as well as non-transfected F50 cells and F50-derived induced pluripotent stem cells.

FIG. 17 is a series of representative microscopy images of adult human primary fibroblasts expanded from single cells that were not transfected (top two rows) or transfected with compositions of the present disclosure (+hTERT/dCas9−VPR+gRNA; bottom two rows).

FIG. 18 is a schematic overview of the transendothelial migration (TEM) assay.

FIG. 19 is chart showing the migration activity of hMSCs transfected with compositions of the present disclosure as measured using the TEM assay.

FIG. 20 is series chart showing the oxidation level of thiol groups detected in selected proteins in senescent hMSCs transfected with compositions of the present disclosure as compared to non-transfected young low passage and senescent high passage hMSCs.

FIG. 21 is a series chart showing the degree of methylation at 9 senescence-associated DNA methylation sites in senescent cells of different types transfected with compositions of the present disclosure as compared to non-transfected young low passage and senescent high passage cells.

DETAILED DESCRIPTION OF THE INVENTION

Telomeres comprise repetitive DNA sequences at the ends of linear chromosomes that, when sufficiently long, allow each chromosome end to form a loop that protects the ends from acting as double-stranded or single-stranded DNA breaks. Telomeres shorten over time, due in part to oxidative damage and incomplete DNA replication, eventually leading to critically short telomeres unable to form the protective loop, exposure of the chromosome ends, chromosome-chromosome fusions, DNA damage responses, and cellular senescence, apoptosis, or malignancy.

The enzyme complex telomerase extends telomeres and comprises two essential components: the telomerase reverse transcriptase (TERT), and an RNA component known as telomerase RNA component (TERC). Other components of the telomerase complex include the proteins TCAB1, Dyskerin, Gar1, Nhp2, Nop 10, and RHAU.

Due to the importance of telomere length maintenance in preventing cellular senescence and apoptosis and resulting cellular dysfunction, genetic mutations of TERT and TERC are linked to fatal inherited diseases of inadequate telomere maintenance, including forms of idiopathic pulmonary fibrosis, dyskeratosis congenita, and aplastic anemia. The effects of premature cellular senescence and apoptosis due to short telomeres in these diseases are devastating in themselves, and may be compounded by increased risk of cancer. Moreover, the shortening of telomeres in cells that are cultured in vitro results is also a major problem in the production of therapeutic cell populations, the creation of in vitro synthetic tissue and tumors and the in vitro creation of non-cancerous somatic cells lines for research and drug testing. Repeated passaging in vitro can lead to senescence and the lack of further expansion ability, and in the case of therapeutic cell populations, a decrease in clinically-relevant biological activity.

Thus, there is a clear need in the art for compositions, kits and methods directed to elongating telomeres in order to rejuvenate cells. Existing approaches directed to increasing the expression of TERT and/or TERC in target cells has relied on the use of integrating viruses to obtain the desired increase in TERT and/or TERC expression. However, these approaches suffer from safety concerns, as the integrating viruses can result in potentially dangerous, permanent genome modifications. Moreover, the sustained overexpression of TERT and/or TERC, and concomitant increases in telomere length, have been linked to cancer cell immortalization, making the integrating virus approach dangerous in a clinical context.

Without wishing to be bound by theory, the compositions, kits and methods of the present disclosure allow for the transient increase in TERT and/or TERC expression for a time period that is long enough to rejuvenate the target cells, but short enough to avoid deleterious and dangerous off-target effects. The use of non-integrating RNA molecules in the present disclosure allows for fine-tuning of the expression levels and stoichiometry of rejuvenating factors in a clinically safe manner.

The compositions, kits and methods of the present disclosure can be used for a variety of different research and clinical applications, including, but not limited to, the production of therapeutic cell populations (e.g. CAR-T cell populations, mesenchymal stem/stromal cell populations), the production of in vitro tissue and organs for subsequent transplantation, research or drug testing, the production of genome-edited cell populations for therapeutic and research applications, the rejuvenation of senescent, aged and disease associated cell lines, etc.

Various compositions, kits and methods of the present disclosure are described in full detail herein.

Rejuvenating Compositions

In some aspects, the present disclosure provides a composition comprising: a) at least one first polynucleotide molecule comprising a nucleic acid sequence encoding at least a portion of telomerase reverse transcriptase (TERT); and b) at least one second polynucleotide molecule comprising a nucleic acid sequence encoding at least a portion of at least one DNA targeting polypeptide, wherein the DNA targeting polypeptide increases transcription of telomerase RNA component (TERC).

In some aspects, the present disclosure provides a composition comprising: a) at least one first polynucleotide molecule comprising a nucleic acid sequence encoding at least a portion of telomerase reverse transcriptase (TERT); and b) at least one DNA targeting polypeptide, wherein the DNA targeting polypeptide increases transcription of telomerase RNA component (TERC).

In some aspects, the at least one first polynucleotide molecule comprising a nucleic acid sequence encoding at least a portion of telomerase reverse transcriptase (TERT) can be an mRNA molecule encoding at least a portion of TERT. In some aspects, the at least one first polynucleotide molecule can be a plasmid comprising a nucleic acid sequence encoding at least a portion of TERT operably linked to at least one promoter sufficient to drive expression of the at least one portion of TERT.

In some aspects, the at least one second polynucleotide molecule comprising a nucleic acid sequence encoding at least a portion of at least one DNA targeting polypeptide can be an mRNA molecule encoding at least a portion of at least one DNA targeting polypeptide. In some aspects, the at least one second polynucleotide molecule can be a plasmid comprising a nucleic acid sequence encoding at least a portion of at least one DNA targeting polypeptide operably linked to at least one promoter sufficient to drive expression of the at least one portion of the at least one DNA targeting polypeptide.

Thus, the present disclosure provides a composition comprising: a) at least one first mRNA molecule encoding at least a portion of telomerase reverse transcriptase (TERT); and b) at least one second mRNA molecule encoding at least a portion of at least one DNA targeting polypeptide, wherein the DNA targeting polypeptide increases transcription of telomerase RNA component (TERC).

The present disclosure also provides a composition comprising: a) at least one plasmid comprising a nucleic acid sequence encoding at least a portion of TERT operably linked to at least one promoter sufficient to drive expression of the at least one portion of TERT; and b) at least one second mRNA molecule encoding at least a portion of at least one DNA targeting polypeptide, wherein the DNA targeting polypeptide increases transcription of telomerase RNA component (TERC).

The present disclosure also provides a composition comprising: a) at least one first mRNA molecule encoding at least a portion of telomerase reverse transcriptase (TERT); and b) at least one plasmid comprising a nucleic acid sequence encoding at least a portion of at least one DNA targeting polypeptide operably linked to at least one promoter sufficient to drive expression of the at least one portion of the at least one DNA targeting polypeptide, wherein the DNA targeting polypeptide increases transcription of telomerase RNA component (TERC).

The present disclosure also provides a composition comprising: a) at least one plasmid comprising a nucleic acid sequence encoding at least a portion of TERT operably linked to at least one promoter sufficient to drive expression of the at least one portion of TERT; and b) at least one plasmid comprising a nucleic acid sequence encoding at least a portion of at least one DNA targeting polypeptide operably linked to at least one promoter sufficient to drive expression of the at least one portion of the at least one DNA targeting polypeptide, wherein the DNA targeting polypeptide increases transcription of telomerase RNA component (TERC).

In some aspects, a DNA targeting polypeptide can comprise at least one Cas9 molecule, at least one Cas9 variant molecule, at least one Cas9 ortholog molecule or any combination thereof.

In some aspects, a Cas9 molecule, a Cas9 variant molecule or a Cas9 ortholog molecule can be nuclease-deficient or nuclease-dead. As used herein, the term “dCas9” is used in its broadest sense to refer to a Cas9 molecule, ortholog and/or variant that is nuclease-deficient or nuclease dead. In a non-limiting example, a Cas9 molecule, a Cas9 variant molecule or a Cas9 ortholog molecule can comprise at least one mutation, deletion or insertion which renders the Cas9 molecule, the Cas9 variant molecule or the Cas9 ortholog molecule nuclease-deficient or nuclease-dead.

In some aspects, a Cas9 variant molecule can comprise eSpCas9 (K855A), eSpCas9 (1.0), eSpCas9 (1.1), SpCas9-HF1 (VP12), HypaCas9, xCas9, SpyFi Cas9, iSpy Cas9, iSpyMac, Cas9 (VQR), Cas9 (EQR), Cas9 (VRER), Cas9 (D1135E), Cas9(QQR1), SaCas9 (KKH), Nme1 Cas9, Nme2Cas9, Nme3Cas9 or any combination thereof.

In some aspects, a Cas9 ortholog molecule can comprise Streptococcus pyogenes Cas9 (spCas9), Francisella novicida Cas9 (FnCas9), Staphylococcus aureus Cas9 (SaCas9), Neisseria meningitidis Cas9 (NmCas9; NmeCas9), Streptococcus thermophilus CRISPR1-Cas9 (St1Cas9), Streptococcus thermophilus CRISPR3-Cas9 (St3Cas9), Campylobacter jejuni Cas9 (CjCas9), Acidaminococcus sp. BV3L6 Cpf1 (AsCpf1), Lachnospiraceae bacterium ND2006 Cpf1 (LbCpf1), Streptococcus canis Cas9 (ScCas9), Treponema denticola Cas9 (TdCas9), Streptococcus macacae Cas9 (SmacCas9), Casϕ (Cas12j), Francisella tularensis subsp. novicida Cas9, Pasteurella multocida Cas9, Campylobacter lari CF89-12 Cas9, Mycoplasma gallisepticum str. F Cas9, Nitratifractor salsuginis str DSM 16511 Cas9, Parvibaculum lavamentivorans Cas9, Roseburia intestinalis Cas9, Neisseria cinerea Cas9, Gluconacetobacter diazotrophicus Cas9, Azospirillum B510 Cas9, Sphaerochaeta globus str. Buddy Cas9, Flavobacterium columnare Cas9, Fluviicola taffensis Cas9, Bacteroides coprophilus Cas9, Mycoplasma mobile Cas9, Lactobacillus farciminis Cas9, Streptococcus pasteurianus Cas9, Lactobacillus johnsonii Cas9, Staphylococcus pseudintermedius Cas9, Filifactor alocis Cas9, Legionella pneumophila str. Paris Cas9, Sutterella wadsworthensis Cas9, Corynebacter diphtheriae Cas9 or any combination thereof.

In some aspects, a Cas9 ortholog molecule can comprise a chimeric variant of Streptococcus pyogenes Cas9 (spCas9), Francisella novicida Cas9 (FnCas9), Staphylococcus aureus Cas9 (SaCas9), Neisseria meningitidis Cas9 (NmCas9; NmeCas9), Streptococcus thermophilus CRISPR1-Cas9 (St1Cas9), Streptococcus thermophilus CRISPR3-Cas9 (St3Cas9), Campylobacter jejuni Cas9 (CjCas9), Acidaminococcus sp. BV3L6 Cpf1 (AsCpf1), Lachnospiraceae bacterium ND2006 Cpf1 (LbCpf1), Streptococcus canis Cas9 (ScCas9), Treponema denticola Cas9 (TdCas9), Streptococcus macacae Cas9 (SmacCas9), Casϕ (Cas12j), Francisella tularensis subsp. novicida Cas9, Pasteurella multocida Cas9, Campylobacter lari CF89-12 Cas9, Mycoplasma gallisepticum str. F Cas9, Nitratifractor salsuginis str DSM 16511 Cas9, Parvibaculum lavamentivorans Cas9, Roseburia intestinalis Cas9, Neisseria cinerea Cas9, Gluconacetobacter diazotrophicus Cas9, Azospirillum B510 Cas9, Sphaerochaeta globus str. Buddy Cas9, Flavobacterium columnare Cas9, Fluviicola taffensis Cas9, Bacteroides coprophilus Cas9, Mycoplasma mobile Cas9, Lactobacillus farciminis Cas9, Streptococcus pasteurianus Cas9, Lactobacillus johnsonii Cas9, Staphylococcus pseudintermedius Cas9, Filifactor alocis Cas9, Legionella pneumophila str. Paris Cas9, Sutterella wadsworthensis Cas9, Corynebacter diphtheriae Cas9 or any combination thereof.

In some aspects, a DNA targeting polypeptide can comprise at least one TALE molecule, at least one zinc-finger molecule, at least one meganuclease molecule or any combination thereof.

In some aspects, a DNA targeting polypeptide can comprise at least one transactivation molecule. In some aspects, a transactivation molecule is a molecule that binds to transcription factors and/or transcriptional co-regulators that are capable of driving transcription of a target gene.

In some aspects, a transactivation molecule can comprise at least one P65 molecule, at least one Rta molecule, at least one VP16 molecule, at least one VP64 molecule, at least one VP160 molecule, at least one VP64-P65-Rta (VPR) molecule, at least one SunTag peptide, at least one single guide RNA-MS2 (sgRNA-MS2) molecule or any combination thereof.

In some aspects, a DNA targeting polypeptide can be a DNA targeting ribonucleoprotein (RNP) complex. A DNA targeting ribonucleoprotein complex can comprise both at least one protein component and at least one nucleic acid component. The at least one protein component can comprise any of the protein components described herein, including, but not limited to, a transactivation molecule, a Cas9 molecule, a Cas9 variant molecule or a Cas9 ortholog molecule, a TALE molecule, a zinc-finger molecule, a meganuclease molecule or any combination thereof. The at least one nucleic acid component can be a ribonucleic acid component. The at least one nucleic acid component can comprise any of the nucleic acid components described herein, including, but not limited to, a guide RNA molecule, a single guide RNA molecule, a single guide RNA-MS2 (sgRNA-MS2) molecule or any combination thereof.

In some aspects, a DNA targeting polypeptide can further comprise at least one cell-penetrating peptide. A cell-penetrating peptide can comprise at least a portion of an HIV-derived TAT protein, polyarginine, any other cell-penetrating peptide known in the art or any combination thereof.

In some aspects, a DNA targeting polypeptide can comprise at least one guide RNA. In some aspects, a transactivation molecule can comprise at least one single guide RNA-MS2 (sgRNA-MS2) molecule. In some aspects, a sgRNA-MS2 molecule can comprise a nucleic acid sequence complementary to a nucleic acid sequence located upstream, within, or downstream of the endogenous TERC gene and at least about one, or at least about two, or at least about three, or at least about four, or at least about five, or at least about six, or at least about seven, or at least about eight, or at least about nine, or at least about ten MS2 RNA aptamers.

In some aspects, a DNA targeting polypeptide can comprise a dCas9 molecule and a VPR molecule.

In some aspects, a DNA targeting polypeptide can bind upstream of, 5′ to, within, downstream of or 3′ to the endogenous TERC gene, e.g. the endogenous human TERC gene.

In some aspects, an at least one DNA targeting polypeptide can bind at least about 0.1 kilobases (kb), or at least about 0.5 kb, or at least about 1.0 kb, or at least about 1.5 kb, or at least about 2.0 kb, or at least about 2.5 kb, or at least about 3.0 kb, or at least about 3.5 kb, or at least about 4.0 kb, or at least about 4.5 kb, or at least about 5.0 kb, or at least about 5.5 kb, or at least about 6.0 kb, or at least about 6.5 kb, or at least about 7.0 kb, or at least about 7.5 kb, or at least about 8.5 kb, or at least about 9.0 kb, or at least about 9.5 kb, or at least about 10.0 kb, or at least about 15 kb, or at least about 20 kb, or at least about 30 kb, or at least about 40 kb, or at least about 50 kb, or at least about 60 kb, or at least about 15 kb, or at least about 70 kb, or at least about 80 kb, or at least about 90 kb, or at least about 100 kb, or at least about 250 kb, or at least about 500 kb, or at least about 750 kb, or at least about 1000 kb, or at least 5000 kb or at least about 10,000 kb upstream of the endogenous TERC gene, e.g. the endogenous human TERC gene.

In some aspects, an at least one DNA targeting polypeptide can bind at least about 0.1 kilobases (kb), or at least about 0.5 kb, or at least about 1.0 kb, or at least about 1.5 kb, or at least about 2.0 kb, or at least about 2.5 kb, or at least about 3.0 kb, or at least about 3.5 kb, or at least about 4.0 kb, or at least about 4.5 kb, or at least about 5.0 kb, or at least about 5.5 kb, or at least about 6.0 kb, or at least about 6.5 kb, or at least about 7.0 kb, or at least about 7.5 kb, or at least about 8.5 kb, or at least about 9.0 kb, or at least about 9.5 kb, or at least about 10.0 kb, or at least about 15 kb, or at least about 20 kb, or at least about 30 kb, or at least about 40 kb, or at least about 50 kb, or at least about 60 kb, or at least about 15 kb, or at least about 70 kb, or at least about 80 kb, or at least about 90 kb, or at least about 100 kb, or at least about 250 kb, or at least about 500 kb, or at least about 750 kb, or at least about 1000 kb, or at least 5000 kb or at least about 10,000 kb 3′ to the endogenous TERC gene, e.g. the endogenous human TERC gene.

In some aspects, an at least one DNA targeting polypeptide can bind at least about 0.1 kilobases (kb), or at least about 0.5 kb, or at least about 1.0 kb, or at least about 1.5 kb, or at least about 2.0 kb, or at least about 2.5 kb, or at least about 3.0 kb, or at least about 3.5 kb, or at least about 4.0 kb, or at least about 4.5 kb, or at least about 5.0 kb, or at least about 5.5 kb, or at least about 6.0 kb, or at least about 6.5 kb, or at least about 7.0 kb, or at least about 7.5 kb, or at least about 8.5 kb, or at least about 9.0 kb, or at least about 9.5 kb, or at least about 10.0 kb, or at least about 15 kb, or at least about 20 kb, or at least about 30 kb, or at least about 40 kb, or at least about 50 kb, or at least about 60 kb, or at least about 15 kb, or at least about 70 kb, or at least about 80 kb, or at least about 90 kb, or at least about 100 kb, or at least about 250 kb, or at least about 500 kb, or at least about 750 kb, or at least about 1000 kb, or at least 5000 kb or at least about 10,000 kb downstream of the endogenous TERC gene, e.g. the endogenous human TERC gene.

In some aspects, an at least one DNA targeting polypeptide can bind at least about 0.1 kilobases (kb), or at least about 0.5 kb, or at least about 1.0 kb, or at least about 1.5 kb, or at least about 2.0 kb, or at least about 2.5 kb, or at least about 3.0 kb, or at least about 3.5 kb, or at least about 4.0 kb, or at least about 4.5 kb, or at least about 5.0 kb, or at least about 5.5 kb, or at least about 6.0 kb, or at least about 6.5 kb, or at least about 7.0 kb, or at least about 7.5 kb, or at least about 8.5 kb, or at least about 9.0 kb, or at least about 9.5 kb, or at least about 10.0 kb, or at least about 15 kb, or at least about 20 kb, or at least about 30 kb, or at least about 40 kb, or at least about 50 kb, or at least about 60 kb, or at least about 15 kb, or at least about 70 kb, or at least about 80 kb, or at least about 90 kb, or at least about 100 kb, or at least about 250 kb, or at least about 500 kb, or at least about 750 kb, or at least about 1000 kb, or at least 5000 kb or at least about 10,000 kb 5′ to the endogenous TERC gene, e.g. the endogenous human TERC gene.

In some aspects, an mRNA molecule of any composition of the present disclosure can be a modified mRNA molecule.

In some aspects, a modified mRNA molecule can comprise at least one modified ribonucleoside base. A modified ribonucleoside base can comprise a pseudouridine (Ψ) residue, a 5-methylcytidine (m⁵C) residue or any combination thereof.

In some aspects, a modified mRNA molecule can comprise at least one modified nucleoside. A modified nucleoside can comprise 5-methylcytidine (m⁵C), 5-methyluridine (m⁵U), N6-methyladenosine (m⁶A), inosine and 2′-0-methylated nucleosides, in addition to N7-methylguanosine (m⁷G), 2-thiouridine (s²U), pseudouridine (ψ), 2′-0-methyl-U, m¹A (1-methyladenosine); m²A (2-methyladenosine); Am (2′-0-methyladenosine); ms²m⁶A (2-methylthio-N⁶-methyladenosine); i⁶A (N⁶-isopentenyladenosine); ms²i6A (2-methylthio-N⁶isopentenyladenosine); io⁶A (N⁶-(cis-hydroxyisopentenyl)adenosine); ms²i⁶A (2-methylthio-N⁶-(cis-hydroxyisopentenyl)adenosine); g⁶A (N⁶-glycinylcarbamoyladenosine); t⁶A (N⁶-threonylcarbamoyladenosine); ms²t⁶A (2-methylthio-N⁶-threonyl carbamoyladenosine); m⁶t⁶A (N⁶-methyl-N⁶-threonylcarbamoyladenosine); hn⁶A(N⁶-hydroxynorvalylcarbamoyladenosine); ms²hn⁶A (2-methylthio-N⁶-hydroxynorvalyl carbamoyladenosine); Ar(p) (2′-0-ribosyladenosine(phosphate)); I (inosine); m¹I (1-methylinosine); m¹Im (1,2′-0-dimethylinosine); m³C (3-methylcytidine); Cm (2′-0-methylcytidine); s²C (2-thiocytidine); ac⁴C(N⁴-acetylcytidine); f⁵C (5-formylcytidine); m⁵ Cm (5,2′-0-dimethylcytidine); ac⁴Cm (N⁴-acetyl-2′-0-methylcytidine); k²C (lysidine); m¹G (1-methylguanosine); m²G (N²-methylguanosine); m⁷G (7-methylguanosine); Gm (2′-0-methylguanosine); m² ₂G (N²,N²-dimethylguanosine); m²Gm (N²,2′-0-dimethylguanosine); m² ₂Gm (N²,N²,2′-0-trimethylguanosine); Gr(p) (2′-0-ribosylguanosine (phosphate)); yW (wybutosine); o₂yW (peroxywybutosine); OHyW (hydroxywybutosine); OHyW* (undermodified hydroxywybutosine); imG (wyosine); mimG (methylwyosine); Q (queuosine); oQ (epoxyqueuosine); galQ (galactosyl-queuosine); manQ (mannosyl-queuosine); preQ₀ (7-cyano-7-deazaguanosine); preQ₁ (7-aminomethyl-7-deazaguanosine); G⁺ (archaeosine); D (dihydrouridine); m⁵Um (5,2′-0-dimethyluridine); s⁴U (4-thiouridine); m⁵s²U (5-methyl-2-thiouridine); s²Um (2-thio-2′-0-methyluridine); acp³U (3-(3-amino-3-carboxypropyl)uridine); ho⁵U (5-hydroxyuridine); mo⁵U (5-methoxyuridine); cmo⁵U (uridine 5-oxyacetic acid); mcmo⁵U (uridine 5-oxyacetic acid methyl ester); chm⁵U (5-(carboxyhydroxymethyl)uridine)); mchm⁵U (5-(carboxyhydroxymethyl)uridine methyl ester); mcm⁵U (5-methoxycarbonylmethyluridine); mcm⁵Um (5-methoxycarbonylmethyl-2′-0-methyluridine); mcm⁵s²U (5-methoxycarbonylmethyl-2-thiouridine); nm⁵s²U (5-aminomethyl-2-thiouridine); mnm⁵U (5-methylaminomethyluridine); mnm⁵s²U (5-methylaminomethyl-2-thiouridine); mnm⁵se²U (5-methylaminomethyl-2-selenouridine); ncm⁵U (5-carbamoylmethyluridine); ncm⁵Um (5-carbamoylmethyl-2′-0-methyluridine); cmnm⁵U (5-carboxymethylaminomethyluridine); cmnm⁵Um (5-carboxymethylaminomethyl-2′-0-methyluridine); cmnm⁵s²U (5-carboxymethylaminomethyl-2-thiouridine); m⁶ ₂A (N⁶,N⁶-dimethyladenosine); Im (2′-0-methylinosine); m⁴C(N⁴-methylcytidine); m⁴ Cm (N⁴,2′-0-dimethylcytidine); hm⁵C (5-hydroxymethylcytidine); m³U (3-methyluridine); cm⁵U (5-carboxymethyluridine); m⁶Am (N⁶,2′-0-dimethyladenosine); m⁶ ₂Am (N⁶,N⁶,0-2′- 2 7 2 2 2 7 2 2trimethyladenosine); m^(2,7)G (N²,7-dimethylguanosine); m^(2,2,7)G (N², N²,7-trimethylguanosine); m³Um (3,2′-0-dimethyluridine); m⁵D (5-methyldihydrouridine); f⁵Cm (5-formyl-2′-0-methylcytidine); m¹Gm (1,2′-0-dimethylguanosine); m¹Am (1,2′-0-dimethyladenosine); τm⁵U (5-taurinomethyluridine); τm⁵s²U (5-taurinomethyl-2-thiouridine)); imG-14 (4-demethylwyosine); imG2 (isowyosine); ac⁶A (N⁶-acetyladenosine), or any combination thereof.

In some aspects, an mRNA molecule can be chemically synthesized using methods standard in the art. In some aspects, an mRNA molecule can be chemically synthesized such that the mRNA molecule comprises at least one chemical modification. In some aspects, an mRNA molecule can be produced by in vitro transcription methods standard in the art, including, but not limited to, in vitro transcription using a plasmid template, in vitro transcription using a PCR-based template. In some aspects, in vitro transcription methods can be performed such that the produced mRNA molecules comprise at least one chemical modification.

In some aspects, a purified DNA targeting polypeptide can be produced using methods standard in the art, including, but not limited to, recombinant protein expression and purification in a bacterial, fungal, insect and/or mammalian system, ion-exchange chromatography, affinity chromatography, immunoaffinity chromatography, size exclusion chromatography, and/or other standard protein production/purification methods known in the art.

In some aspects, a purified DNA-targeting ribonucleoprotein (RNP) complex can be produced using methods standard in the art, including, but not limited to recombinant protein expression and purification in a bacterial, fungal, insect and/or mammalian system, in vitro RNA transcription, ion-exchange chromatography, affinity chromatography, immunoaffinity chromatography, size exclusion chromatography, other standard protein production/purification methods known in the art, and/or other standard nucleic acid production/purification methods known in the art. In some aspects, a preassembled RNP complex that comprises both at least one protein component and at least one nucleic acid can be assembled in vivo (i.e. in a bacterial, fungal, insect and/or mammalian recombinant expression system) and co-purified. In some aspects, a RNP complex can be assembled in vitro after the individual purification of the at least one protein component and the at least one nucleic acid component.

In some aspects, any of the compositions of the present disclosure can further comprise a plurality of guide RNA (gRNA) molecules, wherein at least one gRNA in the plurality is complementary to a nucleic acid sequence located upstream, within, or downstream of the endogenous TERC gene. In some aspects, a plurality of gRNA molecules can comprise at least about one, or at least about two, or at least about three, or at least about four, or at least about five, or at least about six, or at least about seven, or at least about eight, or at least about nine, or at least about ten, or at least about 11, or at least about 12, or at least about 13, or at least about 14, or at least about 15, at least about 16, or at least about 17, or about at least 18, or at least about 19, or at least about 20, or at least about 30, or at least about 40, or at least about 50, or at least about 60, or at least about 70, or at least about 80, or at least about 90, or at least about 100, or at least about 500 or at least about 1000 distinct species of gRNA molecules, wherein each species has a different nucleic acid sequence.

In some aspects, any of the compositions of the present disclosure can further comprise at least one plasmid comprising at least one nucleic acid sequence encoding at least one species of gRNA operably linked to at least one promoter sufficient to drive expression of the at least one species gRNA. In some aspects, any of the compositions of the present disclosure can further comprise at least one plasmid comprising at least one nucleic acid sequence encoding least about two, or at least about three, or at least about four, or at least about five, or at least about six, or at least about seven, or at least about eight, or at least about nine, or at least about ten, or at least about 11, or at least about 12, or at least about 13, or at least about 14, or at least about 15, at least about 16, or at least about 17, or about at least 18, or at least about 19, or at least about 20, or at least about 30, or at least about 40, or at least about 50, or at least about 60, or at least about 70, or at least about 80, or at least about 90, or at least about 100, or at least about 500 or at least about 1000 distinct species of gRNA molecules operably linked to at least one promoter sufficient to drive expression of the gRNA species, wherein each species has a different nucleic acid sequence.

In some aspects, a plurality of gRNA molecules can comprise a plurality of single guide RNA (sgRNA) molecules, crRNA:tracrRNA molecules, truncated sgRNA molecules, high fidelity scaffold gRNA molecules or any combination thereof.

In some aspects, a plurality of gRNA molecules can comprise a plurality of single guide RNA (sgRNA) molecules. In some aspects, a sgRNA molecule can comprise a nucleic acid sequence complementary to a nucleic acid sequence located upstream, within, or downstream of the endogenous TERC gene and at least one MS2 RNA aptamer. In some aspects, a sgRNA molecule can comprise at least about two, or at least about three, or at least about four, or at least about five, or at least about six, or at least about seven, or at least about eight, or at least about nine, or at least about ten MS2 RNA aptamers.

In some aspects, a guide RNA molecule of any composition of the present disclosure can be a modified guide RNA (mod gRNA) molecule.

In some aspects, a modified guide RNA can comprise at least one modified ribonucleoside base. A modified ribonucleoside base can comprise a pseudouridine (Ψ) residue, a 5-methylcytidine (m⁵C) residue or any combination thereof.

In some aspects, a modified guide RNA can comprise at least one modified nucleoside. A modified nucleoside can comprise 5-methylcytidine (m⁵C), 5-methyluridine (m⁵U), N6-methyladenosine (m⁶A), inosine and 2′-0-methylated nucleosides, in addition to N7-methylguanosine (m⁷G), 2-thiouridine (s²U), pseudouridine (ψ), 2′-0-methyl-U, m¹A (1-methyladenosine); m²A (2-methyladenosine); Am (2′-0-methyladenosine); ms²m⁶A (2-methylthio-N⁶-methyladenosine); i⁶A (N⁶-isopentenyladenosine); ms²i6A (2-methylthio-N⁶isopentenyladenosine); io⁶A (N⁶-(cis-hydroxyisopentenyl)adenosine); ms²i⁶A (2-methylthio-N⁶-(cis-hydroxyisopentenyl)adenosine); g⁶A (N⁶-glycinylcarbamoyladenosine); t⁶A (N⁶-threonylcarbamoyladenosine); ms²t⁶A (2-methylthio-N⁶-threonyl carbamoyladenosine); m⁶t⁶A (N⁶-methyl-N⁶-threonylcarbamoyladenosine); hn⁶A(N⁶-hydroxynorvalylcarbamoyladenosine); ms²hn⁶A (2-methylthio-N⁶-hydroxynorvalyl carbamoyladenosine); Ar(p) (2′-0-ribosyladenosine(phosphate)); I (inosine); m¹I (1-methylinosine); m¹Im (1,2′-0-dimethylinosine); m³C (3-methylcytidine); Cm (2′-0-methylcytidine); s²C (2-thiocytidine); ac⁴C(N⁴-acetylcytidine); f⁵C (5-formylcytidine); m⁵Cm (5,2′-0-dimethylcytidine); ac⁴Cm (N⁴-acetyl-2′-0-methylcytidine); k²C (lysidine); m¹G (1-methylguanosine); m²G (N²-methylguanosine); m⁷G (7-methylguanosine); Gm (2′-0-methylguanosine); m² ₂G (N²,N²-dimethylguanosine); m²Gm (N²,2′-0-dimethylguanosine); m² ₂Gm (N²,N²,2′-0-trimethylguanosine); Gr(p) (2′-0-ribosylguanosine (phosphate)); yW (wybutosine); o₂yW (peroxywybutosine); OHyW (hydroxywybutosine); OHyW* (undermodified hydroxywybutosine); imG (wyosine); mimG (methylwyosine); Q (queuosine); oQ (epoxyqueuosine); galQ (galactosyl-queuosine); manQ (mannosyl-queuosine); preQ₀ (7-cyano-7-deazaguanosine); preQ₁ (7-aminomethyl-7-deazaguanosine); G⁺ (archaeosine); D (dihydrouridine); m⁵Um (5,2′-0-dimethyluridine); s⁴U (4-thiouridine); m⁵s²U (5-methyl-2-thiouridine); s²Um (2-thio-2′-0-methyluridine); acp³U (3-(3-amino-3 -carboxypropyl)uridine); ho⁵U (5-hydroxyuridine); mo⁵U (5-methoxyuridine); cmo⁵U (uridine 5-oxyacetic acid); mcmo⁵U (uridine 5-oxyacetic acid methyl ester); chm⁵U (5-(carboxyhydroxymethyl)uridine)); mchm⁵U (5-(carboxyhydroxymethyl)uridine methyl ester); mcm⁵U (5-methoxycarbonylmethyluridine); mcm⁵Um (5-methoxycarbonylmethyl-2′-0-methyluridine); mcm⁵s²U (5-methoxycarbonylmethyl-2-thiouridine); nm⁵ _(s) ²U (5-aminomethyl-2-thiouridine); mnm⁵U (5-methylaminomethyluridine); mnm⁵ _(s) ²U (5-methylaminomethyl-2-thiouridine); mnm⁵se²U (5-methylaminomethyl-2-selenouridine); ncm⁵U (5-carbamoylmethyluridine); ncm⁵Um (5-carbamoylmethyl-2′-0-methyluridine); cmnm⁵U (5-carboxymethylaminomethyluridine); cmnm⁵Um (5-carboxymethylaminomethyl-2′-0-methyluridine); cmnm⁵ _(s) ²U (5-carboxymethylaminomethyl-2-thiouridine); m⁶ ₂A (N⁶,N⁶-dimethyladenosine); Im (2′-0-methylinosine); m⁴C(N⁴-methylcytidine); m⁴ Cm (N⁴,2′-0-dimethylcytidine); hm⁵C (5-hydroxymethylcytidine); m³U (3-methyluridine); cm⁵U (5-carboxymethyluridine); m⁶Am (N⁶,2′-0-dimethyladenosine); m⁶ ₂Am (N⁶,N⁶,0-2′- 2 7 2 2 2 7 2 2trimethyladenosine); m^(2,7)G (N²,7-dimethylguanosine); m^(2,2,7)G (N²,N²,7-trimethylguanosine); m³Um (3,2′-0-dimethyluridine); m⁵D (5-methyldihydrouridine); f⁵Cm (5-formyl-2′-0-methylcytidine); m¹Gm (1,2′-0-dimethylguanosine); m¹Am (1,2′-0-dimethyladenosine); τm⁵U (5-taurinomethyluridine); τm⁵ _(s) ²U (5-taurinomethyl-2-thiouridine)); imG-14 (4-demethylwyosine); imG2 (isowyosine); ac⁶A (N⁶-acetyladenosine), or any combination thereof.

In some aspects, a guide RNA molecule can comprise any sequence recited in Table 1 or Table 2.

TABLE Guide RNA Sequences Seq Seq Seq ID Seq ID Name Sequence NO Name Sequence NO 1015rev UGUUCAUAAA 1 1934rev GAGAAGCCCC 265 UUUACUGACA GGGCCGACCG 1025forw AAAAAAAUCG 2 1937forw CGAACCCCGC 266 UUACAAUUUA CUGGAGGCCG 1028forw AAAAUCGUUA 3 1941forw CCCCGCCUGG 267 CAAUUUAUGG AGGCCGCGGU 1037rev UCUUGAUGAG 4 1944rev GGUGCCUCCG 268 GUAAAAAGAG GAGAAGCCCC 1038rev GUCUUGAUGA 5 1945rev GGGUGCCUCC 269 GGUAAAAAGA GGAGAAGCCC 1039rev UGUCUUGAUG 6 1946forw CCUGGAGGCC 270 AGGUAAAAAG GCGGUCGGCC 103rev AAUUUCUCUC 7 1947forw CUGGAGGCCG 271 CUUUGCAUAU CGGUCGGCCC 1049rev AGUAGUGCUG 8 1948forw UGGAGGCCGC 272 UGUCUUGAUG GGUCGGCCCG 1059rev AGGGGACCUA 9 1956rev GCGGUGGCAG 273 CUUAGGUAAU UGGGUGCCUC 1066rev CAAUUCCAGG 10 1957forw CGGUCGGCCC 274 GGACCUACUU GGGGCUUCUC 106forw ACGGAGCGAG 11 1960forw UCGGCCCGGG 275 UCCCCGCGCG GCUUCUCCGG 1073forw UUUUAACCUA 12 1965rev CAACUCUUCG 276 UUACCUAAGU CGGUGGCAGU 1077rev UAUCUGCUAG 13 1966rev CCAACUCUUC 277 ACAAUUCCAG GCGGUGGCAG 1078rev GUAUCUGCUA 14 1986forw CCACUGCCAC 278 GACAAUUCCA CGCGAAGAGU 1079rev UGUAUCUGCU 15 1987forw CACUGCCACC 279 AGACAAUUCC GCGAAGAGUU 1081forw UAUUACCUAA 16 199forw UUUGGAGAAU 280 GUAGGUCCCC AAAUUGAAUG 1098rev UCCCUUUUAU 17 1rev CAGAGCCCAA 281 UAGGAAAGAA CUCUUCGCGG 1107rev GACUGAAUCU 18 203forw GAGAAUAAAU 282 CCCUUUUAUU UGAAUGAGGA 1116forw CGCCUUUCUU 19 203rev CCAUUGCCGG 283 UCCUAAUAAA CGAGGGGUGA 1117forw GCCUUUCUUU 20 206rev AACUGAUCAC 284 CCUAAUAAAA CAAAUCUCCA 1129rev CUACUACAUU 21 207rev UAACUGAUCA 285 AUUAAUCUUA CCAAAUCUCC 1139rev CCAGCAACAG 22 209forw AAAUUGAAUG 286 UGGACUCUAG AGGAAGGCCC 1149rev GAGAACAUUA 23 209rev AAGCCCCCAU 287 CCAGCAACAG UGCCGGCGAG 114forw GCUAAAUAUC 24 210rev CAAGCCCCCA 288 CAAUAUGCAA UUGCCGGCGA 1159forw CCUCUAGAGU 25 211rev ACAAGCCCCC 289 CCACUGUUGC AUUGCCGGCG 1168rev GCCUCUCCUU 26 216rev GGUUCACAAG 290 GAGCAGAGGA CCCCCAUUGC 116rev GGUGCACGUC 27 217forw UGAGGAAGGC 291 CCACAGCUCA CCUGGAGAUU 1172rev UCCAGCCUCU 28 217forw GCGCAUCCGU 292 CCUUGAGCAG CACCCCUCGC 1178forw CUGGUAAUGU 29 223forw CCGUCACCCC 293 UCUCUAAAUA UCGCCGGCAA 117rev GGGUGCACGU 30 224forw CGUCACCCCU 294 CCCACAGCUC CGCCGGCAAU 1182forw UUAAAGCCAU 31 225forw GUCACCCCUC 295 CCUCUGCUCA GCCGGCAAUG 1187forw GCCAUCCUCU 32 226forw UCACCCCUCG 296 GCUCAAGGAG CCGGCAAUGG 1191forw UCCUCUGCUC 33 237rev GCCCAGUCAG 297 AAGGAGAGGC UCAGGUUUGG 1193rev UUCCACAAAA 34 238rev GGCCCAGUCA 298 CCAUGCUGAU GUCAGGUUUG 1197forw GCUCAAGGAG 35 239rev UGGCCCAGUC 299 AGGCUGGAGA AGUCAGGUUU 1203forw AAAUAUUUUU 36 240rev CUGGCCCAGU 300 CCUAUCAGCA CAGUCAGGUU 1207forw AGGCUGGAGA 37 243rev AAGACUUGGC 301 AGGCAUUCUA ACUUUAUAUG 1211forw UUCCUAUCAG 38 245rev GCACACUGGC 302 CAUGGUUUUG CCAGUCAGUC 1213forw GAGAAGGCAU 39 255forw AACCCCCAAA 303 UCUAAGGAGA CCUGACUGAC 1214forw AGAAGGCAUU 40 256forw ACCCCCAAAC 304 CUAAGGAGAA CUGACUGACU 1215forw GAAGGCAUUC 41 257rev AUAAUCUUGA 305 UAAGGAGAAG GUACAAGACU 1216forw AAGGCAUUCU 42 259rev CCUGCCAAUU 306 AAGGAGAAGG UGCAGCACAC 1220forw CAUUCUAAGG 43 275forw UGGGCCAGUG 307 AGAAGGGGGC UGCUGCAAAU 1221forw AUUCUAAGGA 44 279forw CCAGUGUGCU 308 GAAGGGGGCA GCAAAUUGGC 1221forw CAUGGUUUUG 45 287forw UACUCAAGAU 309 UGGAAAAGUA UAUAAGCAAU 1225forw UAAGGAGAAG 46 28rev CCUCGCCCCC 310 GGGGCAGGGU GAGAGACCCG 1232forw AAGGGGGCAG 47 290forw CAAAUUGGCA 311 GGUAGGAACU GGAGACGUGA 1234rev CAAGACUCUA 48 295rev UUCAUUUUGG 312 GACAAGUUCU CCGACUUUGG 1241rev GAAUCUUGUC 49 298rev CCAUUCAUUU 313 UCGGCUCAGU UGGCCGACUU 1242rev AGAAUCUUGU 50 305forw CGUGAAGGCA 314 CUCGGCUCAG CCUCCAAAGU 1250rev ACUACAGCAG 51 308rev GGCUCACUGC 315 AAUCUUGUCU CCAUUCAUUU 1257forw AGAACUUGUC 52 318forw CCAAAGUCGG 316 UAGAGUCUUG CCAAAAUGAA 126forw CGGCGCGAUU 53 319forw CAAAGUCGGC 317 CCCUGAGCUG CAAAAUGAAU 127forw GGCGCGAUUC 54 31forw GAGUUGGGCU 318 CCUGAGCUGU CUGUCAGCCG 1281rev CUUUGUGAAA 55 329rev GGAACGGCUC 319 AUAGAUUCCC CAGGCAACCC 1281rev AGAUCACCUU 56 32forw AGUUGGGCUC 320 GAGUAAACUG UGUCAGCCGC 1283forw CUGCUGUAGU 57 330forw AAAAUGAAUG 321 CAGUGCUGCC GGCAGUGAGC 1284forw UGCUGUAGUC 58 331forw AAAUGAAUGG 322 AGUGCUGCCU GCAGUGAGCC 1295forw AGUAAGCCUC 59 331rev UUUCCCCUUC 323 AGUUUACUCA AUAUCUAAGU 1308rev GUUUUGAUCA 60 332forw AAUGAAUGGG 324 UCACAUUUUU CAGUGAGCCG 1332forw AAAAUGUGAU 61 338rev ACCCACGCAG 325 GAUCAAAACU GAACGGCUCC 1335forw UUCUUCUCUU 62 340forw GGCAGUGAGC 326 UCUUUUGAGA CGGGGUUGCC 1341rev CCAGCUCUGG 63 345rev CGGGAGAACC 327 GUGACAGAGU CACGCAGGAA 1342rev UCCAGCUCUG 64 346forw UAGUGCCUAC 328 GGUGACAGAG UUAGAUAUGA 1353rev GGACACUGCA 65 347forw AGUGCCUACU 329 CUCCAGCUCU UAGAUAUGAA 1354forw GAAUUAGUGU 66 348forw GUGCCUACUU 330 UCUGUGUCUU AGAUAUGAAG 1354rev GGGACACUGC 67 350rev GAAGACGGGA 331 ACUCCAGCUC GAACCCACGC 1357rev GAAUUCACAG 68 356forw UUAGAUAUGA 332 GAAGAUUUUA AGGGGAAAGA 1358rev GGAAUUCACA 69 356forw UGCCUGGAGC 333 GGAAGAUUUU CGUUCCUGCG 1361forw CCCACUCUGU 70 357forw UAGAUAUGAA 334 CACCCAGAGC GGGGAAAGAA 1369rev ACCUUAAAAA 71 357forw GCCUGGAGCC 335 UGGAAUUCAC GUUCCUGCGU 1374rev GGUUGCAGUG 72 364rev GGCAACAAAA 336 AGCCAAGAUG AGCGGAAGAC 1375rev AGGUUGCAGU 73 365rev AGGCAACAAA 337 GAGCCAAGAU AAGCGGAAGA 1376rev GAGGUUGCAG 74 372forw AAGAAGGGUU 338 UGAGCCAAGA UGAGAUAAUG 1379rev CACCUCGACU 75 372rev CCAUAAAAGG 339 ACCUUAAAAA CAACAAAAAG 137rev GCAUGUGUGA 76 373forw AGAAGGGUUU 340 GCCGAGUCCU GAGAUAAUGU 1382forw GGAGUGCAGU 77 385rev AGUUGUAAUA 341 GUCCCCAUCU CAACCAUAAA 1388forw UCCUGUGAAU 78 388forw AAUGUGGGAU 342 UCCAUUUUUA GCUAAGAGAA 138rev UGCAUGUGUG 79 391forw GUGGGAUGCU 343 AGCCGAGUCC AAGAGAAUGG 1395rev GCUAGAAACC 80 392forw CCGCUUUUUG 344 GAGGAGGCAG UUGCCUUUUA 1397forw UUCCAUUUUU 81 40forw UCUGUCAGCC 345 AAGGUAGUCG GCGGGUCUCU 1401rev AAAAUCGCUA 82 413rev CUCAACAAAA 346 GAAACCGAGG UCUGCAGAGC 1404rev UAUCCUCUGC 83 41forw CUGUCAGCCG 347 AGACCAGACG CGGGUCUCUC 1404rev GAGAAAAUCG 84 42forw UGUCAGCCGC 348 CUAGAAACCG GGGUCUCUCG 1407forw CACUGCAACC 85 434forw CUGCUCUGCA 349 UCUGCCUCCU GAUUUUGUUG 140forw GAAAUUAAAG 86 439forw UUUAGCAUCU 350 AUUUAAAAGC ACUCUAUGUA 140forw GAGCUGUGGG 87 43forw GUCAGCCGCG 351 ACGUGCACCC GGUCUCUCGG 1411forw UAGUCGAGGU 88 448rev GACUGGUCGA 352 GAACCGCGUC GAUCUACCUU 1421forw GAACCGCGUC 89 449rev GGACUGGUCG 353 UGGUCUGCAG AGAUCUACCU 1431rev UGUAAACCCA 90 452forw UGAGGUUUUU 354 GCUACUUGGG GCUUCUCCCA 1433forw GUCUGCAGAG 91 465rev CCACACCCCG 355 GAUAGAAAAA UUGAGGGGAC 1434rev GCCUGUAAAC 92 470rev UUCUCCCACA 356 CCAGCUACUU CCCCGUUGAG 1435rev UGCCUGUAAA 93 471rev GUUCUCCCAC 357 CCCAGCUACU ACCCCGUUGA 1436rev AACUAACUUG 94 472forw AGUGCAAUAG 358 AGGUAUCAGA UGCUAAAAAC 1437rev AAACUAACUU 95 472rev UGUUCUCCCA 359 GAGGUAUCAG CACCCCGUUG 1444forw CUCUCAGCCU 96 478forw AUCUCGACCA 360 CCCAAGUAGC GUCCCCUCAA 1445forw UCUCAGCCUC 97 479forw UCUCGACCAG 361 CCAAGUAGCU UCCCCUCAAC 1446rev UUAAAGGUGA 98 480forw CUCGACCAGU 362 AACUAACUUG CCCCUCAACG 1453forw UCCCAAGUAG 99 485forw CCAGUCCCCU 363 CUGGGUUUAC CAACGGGGUG 145rev AUCAUAACAU 100 486forw CAGUCCCCUC 364 AGUUUCCUUA AACGGGGUGU 1462rev UUACUUCCGA 101 488rev CCAGGUUGUA 365 CCUUCUUUAA AAGUUUUUUA 1462rev AAAAAAUCAG 102 48forw CCGCGGGUCU 366 CCGGGUAUGG CUCGGGGGCG 1465rev ACAAAAAAAU 103 49forw CGCGGGUCUC 367 CAGCCGGGUA UCGGGGGCGA 146forw UGGGACGUGC 104 4rev UGACAGAGCC 368 ACCCAGGACU CAACUCUUCG 1470rev AAAAUACAAA 105 506rev UUUCUUUCAU 369 AAAAUCAGCC AGCAUCUGCC 1471rev GAAAAUACAA 106 508forw CCGUAAAAAA 370 AAAAAUCAGC CUUUACAACC 1472forw GUUAGUUUCA 107 530forw GCAGAUGCUA 371 CCUUUAAAGA UGAAAGAAAA 1472forw CAGGCACACA 108 531forw CAGAUGCUAU 372 CCACCAUACC GAAAGAAAAA 1476forw GUUUCACCUU 109 532forw AGAUGCUAUG 373 UAAAGAAGGU AAAGAAAAAG 1495rev CCCUUCCGCA 110 536forw GCUAUGAAAG 374 CGUCCGGGAA AAAAAGGGGA 1500rev CGUUGCCCUU ill 537forw CUAUGAAAGA 375 CCGCACGUCC AAAAGGGGAU 1501rev ACGUUGCCCU 112 549forw AAGGGGAUGG 376 UCCGCACGUC GAGAGAGAGA 1502forw UAAAGACGCA 113 54forw GUCUCUCGGG 377 AAGCCUUUCC GGCGAGGGCG 1502forw UUUUGUAUUU 114 552forw GGGAUGGGAG 378 UCAGUAAAGU AGAGAGAAGG 1503forw UUUGUAUUUU 115 553forw GGAUGGGAGA 379 CAGUAAAGUU GAGAGAAGGA 1507forw UAUUUUCAGU 116 561forw UAGAAGAUCU 380 AAAGUUGGGC AAAUGAACAU 150forw AUUUAAAAGC 117 563forw GAGAGAAGGA 381 AGGAGCCAUA GGGAGAGAGA 1510forw CAAAGCCUUU 118 568forw AAGGAGGGAG 382 CCCGGACGUG AGAGAUGGAG 1511forw UUCAGUAAAG 119 569forw AGGAGGGAGA 383 UUGGGCAGGC GAGAUGGAGA 1514forw GCCUUUCCCG 120 574rev CAUAAACCGA 384 GACGUGCGGA UGACCAUUAA 1514rev CACCUGAGGU 121 57forw AACAAGCGCU 385 CAGGAGUUCG AUGACUAGCA 1515forw CCUUUCCCGG 122 581forw UGGAAAUUGU 386 ACGUGCGGAA GUUCCUUUAA 1523rev CGGGCGGAUC 123 588forw UGUGUUCCUU 387 ACCUGAGGUC UAAUGGUCAU 1524rev UCCAUUUCCG 124 597rev AAAAAGAAAC 388 GCCAUGAGGA UUCUAACCUC 1528rev AAGUUCCAUU 125 598forw UUUACUUUUC 389 UCCGGCCAUG UUUCAGAUCG 1528rev AGAAGCGGGC 126 601forw UGGUCAUCGG 390 GGAUCACCUG UUUAUGCCAG 1532forw GGCCUCGAAC 127 602rev CUCCGUGGAG 391 UCCUGACCUC UUGUCGCUGU 1533forw AAGGGCAACG 128 60forw CGGGGGCGAG 392 UCCUUCCUCA GGCGAGGUUC 1536rev GGAAAUUAAA 129 617rev AUUCAGUUAG 393 GUUCCAUUUC AUAAACUCCG 1537forw GCAACGUCCU 130 620forw GACCGACAGC 394 UCCUCAUGGC GACAACUCCA 1539rev CUUUGGGAGG 131 632rev ACUGCUCAAG 395 CAGAAGCGGG GUCAUCGCCA 1542rev GCACUUUGGG 132 635forw UUUUUUGAAA 396 AGGCAGAAGC AAUUAGACCU 1543forw UCCUUCCUCA 133 63rev UCCUCUUCCU 397 UGGCCGGAAA GCGGCCUGAA 1543rev AGCACUUUGG 134 644rev GGGUUAUAUC 398 GAGGCAGAAG CUACUGCUCA 1552rev UGUAAUCCCA 135 655forw UGGCGAUGAC 399 GCACUUUGGG CUUGAGCAGU 1555rev GCCUGUAAUC 136 663rev CAGUUUUACA 400 CCAGCACUUU UAUAAAUGAC 1556rev CGCCUGUAAU 137 664rev UUGGAACGCU 401 CCCAGCACUU AAGCUUGUGG 1557rev GCGGGCUGGU 138 665rev AUUGGAACGC 402 UGGGGGGAAC UAAGCUUGUG 1558rev GGCGGGCUGG 139 666rev UAUUGGAACG 403 UUGGGGGGAA CUAAGCUUGU 1563rev UCUCGGGCGG 140 667rev UUAUUGGAAC 404 GCUGGUUGGG GCUAAGCUUG 1564rev CUCUCGGGCG 141 683rev UAUGCCUAGU 405 GGCUGGUUGG GUUCCGUUAU 1565forw GCUUCUGCCU 142 68forw UGACUAGCAA 406 CCCAAAGUGC GGUUAAGUGA 1565rev UCUCUCGGGC 143 690forw CAAGCUUAGC 407 GGGCUGGUUG GUUCCAAUAA 1566forw CUUCUGCCUC 144 694forw UAUGUAAAAC 408 CCAAAGUGCU UGCACUAUAC 1566rev CUCUCUCGGG 145 697rev CCGGCCGCGA 409 CGGGCUGGUU AUUUUUAUAA 1567rev ACUCUCUCGG 146 699forw CGUUCCAAUA 410 GCGGGCUGGU ACGGAACACU 1571rev AGUCACUCUC 147 69forw GGGCGAGGUU 411 UCGGGCGGGC CAGGCCUUUC 1574forw UCCCAAAGUG 148 713forw CUGGCCAUUA 412 CUGGGAUUAC UAAAAAUUCG 1575rev UGAGAGUCAC 149 714forw ACACUAGGCA 413 UCUCUCGGGC UAAUGAAAGA 1576rev GUGAGAGUCA 150 716rev CAGGUAUGAG 414 CUCUCUCGGG CCACCGCACC 1579rev CUCGUGAGAG 151 717forw CCAUUAUAAA 415 UCACUCUCUC AAUUCGCGGC 1580rev UCUCGUGAGA 152 718forw CAUUAUAAAA 416 GUCACUCUCU AUUCGCGGCC 1583rev GGAUCUUAGU 153 71rev ACUUUAAGCC 417 CCCCGCACGG UUUCAGUCCC 1586rev AAGGGAUCUU 154 723forw UAAAAAUUCG 418 AGUCCCCGCA CGGCCGGGUG 1591forw UACAGGCGUG 155 726forw AAAUUCGCGG 419 AGCCACCGUG CCGGGUGCGG 1592forw ACAGGCGUGA 156 72rev UCGCUCCGUU 420 GCCACCGUGC CCUCUUCCUG 1593forw CAGGCGUGAG 157 731rev AGGGUUGGGG 421 CCACCGUGCG GUGGGGGGUG 1604rev AUUGGCCAAG 158 735rev UCCCAAAGUG 422 CUGACUCUCG CUGGGAUUAC 1604rev GAGUCCCCGC 159 736rev CUGGGAGGGU 423 CCUUGCAAAA UGGGGGUGGG 1605rev GGAGUCCCCG 160 737rev GCUGGGAGGG 424 CCCUUGCAAA UUGGGGGUGG 1614forw GGACUAAGAU 161 738rev GGCUGGGAGG 425 CCCUUUUGCA GUUGGGGGUG 1615forw GACUAAGAUC 162 739rev CGGCUGGGAG 426 CCUUUUGCAA GGUUGGGGGU 1618forw UAAGAUCCCU 163 73forw AGCAAGGUUA 427 UUUGCAAGGG AGUGAAGGCC 1619forw GAGAGCCGCG 164 740rev CCGGCUGGGA 428 AGAGUCAGCU GGGUUGGGGG 1619forw AAGAUCCCUU 165 743rev CUUCGGCCUC 429 UUGCAAGGGC CCAAAGUGCU 1620forw AGAUCCCUUU 166 743rev CUGCCGGCUG 430 UGCAAGGGCG GGAGGGUUGG 1622rev CGGCCGCCGA 167 744rev GCUUCGGCCU 431 CCGCACGGAU CCCAAAGUGC 1626rev AUGCACUUGU 168 744rev ACUGCCGGCU 432 CUGUAGUUCA GGGAGGGUUG 1627rev GGGAGCGGCC 169 745rev GACUGCCGGC 433 GCCGACCGCA UGGGAGGGUU 1632forw GUCAGCUUGG 170 746rev AGACUGCCGG 434 CCAAUCCGUG CUGGGAGGGU 1636forw GCUUGGCCAA 171 74forw GCAAGGUUAA 435 UCCGUGCGGU GUGAAGGCCA 1639forw UGGCCAAUCC 172 750rev UGGGAGACUG 436 GUGCGGUCGG CCGGCUGGGA 1642rev GAGUCGGCUU 173 751rev GUGGGAGACU 437 AUAAAGGGAG GCCGGCUGGG 1647rev CGGGCGAGUC 174 753forw UACCUGUAAU 438 GGCUUAUAAA CCCAGCACUU 1648rev CCGGGCGAGU 175 754forw ACCUGUAAUC 439 CGGCUUAUAA CCAGCACUUU 1658rev CGGUGCGCUG 176 754rev CUUGUGGGAG 440 CCGGGCGAGU ACUGCCGGCU 1665rev GAAGCAAAAG 177 755rev UCUUGUGGGA 441 UACCACUAGA GACUGCCGGC 1666rev CCGCAACCCG 178 757forw UGUAAUCCCA 442 GUGCGCUGCC GCACUUUGGG 1667rev UCCGCAACCC 179 759rev CAAUUCUUGU 443 GGUGCGCUGC GGGAGACUGC 1668forw CCUUUAUAAG 180 760forw CCACCCCCAA 444 CCGACUCGCC CCCUCCCAGC 1673forw UUUGUUCUUA 181 760rev CUCAAGUGAU 445 CUCCAUCUAG CCACCCGCUU 1678rev CAGGCCCACC 182 766forw AGCACUUUGG 446 CUCCGCAACC GAGGCCGAAG 1679forw CGACUCGCCC 183 767forw GCACUUUGGG 447 GGCAGCGCAC AGGCCGAAGC 1680forw GACUCGCCCG 184 769rev AAAUCAGAGC 448 GCAGCGCACC CAAUUCUUGU 1686forw CCCGGCAGCG 185 76forw GUUCAGGCCU 449 CACCGGGUUG UUCAGGCCGC 1689forw GGCAGCGCAC 186 770forw CUUUGGGAGG 450 CGGGUUGCGG CCGAAGCGGG 1689rev CACCACAAAU 187 770rev GAAAUCAGAG 451 GUUGUAAAUG CCAAUUCUUG 1690forw GCAGCGCACC 188 780forw CGGCAGUCUC 452 GGGUUGCGGA CCACAAGAAU 1693forw GCGCACCGGG 189 783rev CAGGCUGGUC 453 UUGCGGAGGG UCGAACGCCA 1694forw CGCACCGGGU 190 784rev CCAGGCUGGU 454 UGCGGAGGGU CUCGAACGCC 1697rev AAAUGGCCAC 191 786forw CGGGUGGAUC 455 CACCCCUCCC ACUUGAGCCC 1699forw CGGGUUGCGG 192 792rev CCAUUAGCUU 456 AGGGUGGGCC AUUUUCUUAA 1700forw GGGUUGCGGA 193 798rev UUUCACCAUG 457 GGGUGGGCCU UUGCCCAGGC 1703forw UUGCGGAGGG 194 802rev GGGGUUUCAC 458 UGGGCCUGGG CAUGUUGCCC 1704forw UGCGGAGGGU 195 804forw CCUGGCGUUC 459 GGGCCUGGGA GAGACCAGCC 1705forw GCGGAGGGUG 196 805forw CUGGCGUUCG 460 GGCCUGGGAG AGACCAGCCU 1707forw CUCCACAUUU 197 812forw CCUUUAAGAA 461 ACAACAUUUG AAUAAGCUAA 1708forw GAGGGUGGGC 198 813forw CGAGACCAGC 462 CUGGGAGGGG CUGGGCAACA 170rev UCGGCGUUCC 199 815rev UUUGUUUCUU 463 CCCCACCAAC UCAACCUAGU 1710forw CACAUUUACA 200 816rev GUUUGUUUCU 464 ACAUUUGUGG UUCAACCUAG 1711forw GGUGGGCCUG 201 821forw AAAUAAGCUA 465 GGAGGGGUGG AUGGCCCACU 1714rev AGUUAGGGUU 202 821rev UGUGUUUUUA 466 AGACAAAAAA GUAGAGACGG 1716forw UACAACAUUU 203 822rev UUGUGUUUUU 467 GUGGUGGUGC AGUAGAGACG 1717forw ACAACAUUUG 204 823rev UUUGUGUUUU 468 UGGUGGUGCA UAGUAGAGAC 1720rev CUGUGGCCAU 205 824rev UUUUGUGUUU 469 UCUUGCUUCA UUAGUAGAGA 1729rev GCCUACGCCC 206 82forw GCCUUUCAGG 470 UUCUCAGUUA CCGCAGGAAG 1730rev CGCCUACGCC 207 839forw CUAGGUUGAA 471 CUUCUCAGUU AGAAACAAAC 1734forw GCAGGGCCGU 208 83forw AGUGAAGGCC 472 GAAGCAAGAA AGGGACUGAA 1737rev AGAAAAACAU 209 842rev GUCGUGAUAA 473 UCCCAGUCUG GUGGGCAGAA 1741forw UUGUCUAACC 210 850rev AUUACCUUGU 474 CUAACUGAGA CGUGAUAAGU 1742forw UGUCUAACCC 211 851rev AAUUACCUUG 475 UAACUGAGAA UCGUGAUAAG 1745forw AAGCAAGAAU 212 853forw CUAAAAACAC 476 GGCCACAGAC AAAAACUAGC 1746forw AGCAAGAAUG 213 854forw UAAAAACACA 477 GCCACAGACU AAAACUAGCU 1748forw ACCCUAACUG 214 859forw ACACAAAAAC 478 AGAAGGGCGU UAGCUGGGCG 1753rev GCGCGCGGGG 215 862forw CAAAAACUAG 479 AGCAAAAGCA CUGGGCGUGG 175forw CAUGCAGUUC 216 866forw AACUAGCUGG 480 GCUUUCCUGU GCGUGGUGGC 1766rev AGCGAGAAAA 217 866forw UCUGCCCACU 481 ACAGCGCGCG UAUCACGACA 1767rev CAGCGAGAAA 218 871rev UCCUGAGUAG 482 AACAGCGCGC CUGGGAUUAC 1768rev UCAGCGAGAA 219 874rev UUGAAGGUAU 483 AAACAGCGCG GGAUUUGGGA 178forw GCAGUUCGCU 220 878rev GGAAUUGAAG 484 UUCCUGUUGG GUAUGGAUUU 1798forw UUUUUCUCGC 221 879rev UCUCAGCCUC 485 UGACUUUCAG CUGAGUAGCU 1799forw UUUUCUCGCU 222 879rev AGGAAUUGAA 486 GACUUUCAGC GGUAUGGAUU 179forw CAGUUCGCUU 223 87forw UCAGGCCGCA 487 UCCUGUUGGU GGAAGAGGAA 1802forw UCUCGCUGAC 224 880rev GUCUCAGCCU 488 UUUCAGCGGG CCUGAGUAGC 180forw AGUUCGCUUU 225 885rev AUCCUAAGGA 489 CCUGUUGGUG AUUGAAGGUA 1810rev CGGUGGAAGG 226 890forw GCCUGUAAUC 490 CGGCAGGCCG CCAGCUACUC 1813forw UUCAGCGGGC 227 890rev AGAUGAUCCU 491 GGAAAAGCCU AAGGAAUUGA 1816rev AAUGAACGGU 228 893forw UGUAAUCCCA 492 GGAAGGCGGC GCUACUCAGG 181forw UUAUGAUGAA 229 899rev ACUACCCCCA 493 UGUGAUAGUU GAUGAUCCUA 181forw GUUCGCUUUC 230 903forw AUCCAUACCU 494 CUGUUGGUGG UCAAUUCCUU 1820rev CUAGAAUGAA 231 912forw UUCAAUUCCU 495 CGGUGGAAGG UAGGAUCAUC 1823rev GCUCUAGAAU 232 913forw UCAAUUCCUU 496 GAACGGUGGA AGGAUCAUCU 1827rev GUUUGCUCUA 233 914forw CAAUUCCUUA 497 GAAUGAACGG GGAUCAUCUG 182forw UUCGCUUUCC 234 915forw AAUUCCUUAG 498 UGUUGGUGGG GAUCAUCUGG 1830rev UUUGUUUGCU 235 919rev CACUGCAACC 499 CUAGAAUGAA UCUGCCUCCC 1866forw AAACAAAAAA 236 920rev UCACUGCAAC 500 UGUCAGCUGC CUCUGCCUCC 1869rev GGUCCCCGGG 237 921forw ACACGAGAAU 501 AGGGGCGAAC CGCUUGAACC 1870rev AGGUCCCCGG 238 922forw CACGAGAAUC 502 GAGGGGCGAA GCUUGAACCC 1877rev CCGCCGCAGG 239 924rev CCCCUGGCUG 503 UCCCCGGGAG CUCUCUCUCU 1878rev CCCGCCGCAG 240 925forw GAGAAUCGCU 504 GUCCCCGGGA UGAACCCGGG 1879rev ACCCGCCGCA 241 931forw CGCUUGAACC 505 GGUCCCCGGG CGGGAGGCAG 1882rev GCGACCCGCC 242 940rev GGCCUUUAUA 506 GCAGGUCCCC UACACACCCC 1883rev GGCGACCCGC 243 942forw UGCCAAGAGA 507 CGCAGGUCCC GAGAGCAGCC 1884forw GCUGGCCCGU 244 943forw GCCAAGAGAG 508 UCGCCCCUCC AGAGCAGCCA 1885forw CUGGCCCGUU 245 944forw CCAAGAGAGA 509 CGCCCCUCCC GAGCAGCCAG 1886forw UGGCCCGUUC 246 944rev GGAGUCUAGU 510 GCCCCUCCCG GGCGUGAUCU 1890rev CUGGGCAGGC 247 955rev CCAGGCUGGA 511 GACCCGCCGC UGGAGUCUAG 1894forw UCGCCCCUCC 248 958forw AGCCAGGGGU 512 CGGGGACCUG GUGUAUAUAA 1897forw CCCCUCCCGG 249 961rev CAGGCUAUCA 513 GGACCUGCGG CCCUAAAGGU 1898forw CCCUCCCGGG 250 962rev UCAGGCUAUC 514 GACCUGCGGC ACCCUAAAGG 189rev GGGUGACGGA 251 965rev GCUCUUUCGC 515 UGCGCACGAU CCAGGCUGGA 1904rev GCGGGGUUCG 252 965rev GAUUCAGGCU 516 GGGGCUGGGC AUCACCCUAA 1908rev CCAGGCGGGG 253 969rev UCUUGCUCUU 517 UUCGGGGGCU UCGCCCAGGC 1909rev UCCAGGCGGG 254 970forw GUAUAUAAAG 518 GUUCGGGGGC GCCCACCUUU 1913rev GGCCUCCAGG 255 971forw UAUAUAAAGG 519 CGGGGUUCGG CCCACCUUUA 1914rev CGGCCUCCAG 256 973rev GGAGUCUUGC 520 GCGGGGUUCG UCUUUCGCCC 1915rev GCGGCCUCCA 257 975forw CCACUAGACU 521 GGCGGGGUUC CCAUCCAGCC 1916rev CGCGGCCUCC 258 976forw CACUAGACUC 522 AGGCGGGGUU CAUCCAGCCU 1921rev CCGACCGCGG 259 97rev AGGGAAUCGC 523 CCUCCAGGCG GCCGCGCGCG 1922rev GCCGACCGCG 260 980rev ACUUUCAAUC 524 GCCUCCAGGC AUCAGGAUUC 1923rev GGCCGACCGC 261 987rev ACUUCUGACU 525 GGCCUCCAGG UUCAAUCAUC 1926rev CCGGGCCGAC 262 98rev CAGGGAAUCG 526 CGCGGCCUCC CGCCGCGCGC 1928forw CCCAGCCCCC 263 994rev AACGAUUUUU 527 GAACCCCGCC UUUUUUGAGA 1931forw AGCCCCCGAA 264 99rev UCAGGGAAUC 528 CCCCGCCUGG GCGCCGCGCG

TABLE 2 Guide RNA Sequences Seq Seq Seq ID Seq ID Name Sequence NO Name Sequence NO 1420forw GUGAACCGCG 529 967rev AGUCUUGCUC 584 UCUGGUCUGC UUUCGCCCAG A G 1591rev GCUGACUCUC 530 991rev UAACGAUUUU 585 GCGGCUCUCG UUUUUUUGAG U A 634forw AACUCCACGG 531 94rev GCCCAACUCU 586 AGUUUAUCUA UCGCGGUGGC A A 640forw ACGGAGUUUA 532 302rev CCCCAUUGCC 587 UCUAACUGAA GGCGAGGGGU U G 1688forw CCGGCAGCGC 533 997rev AACUACCCCC 588 ACCGGGUUGC AGAUGAUCCU G A 716forw GGCCAUUAUA 534 237rev ACUGCAUGUG 589 AAAAUUCGCG UGAGCCGAGU G C 1608forw AGUGACUCUC 535 309rev CACAAGCCCC 590 ACGAGAGCCG CAUUGCCGGC C G 1678forw GCCGACUCGC 536 579forw GAUCUCGACC 591 CCGGCAGCGC AGUCCCCUCA A A 1728rev GGCGCCUACG 537 HOforw GAGGCACCCA 592 CCCUUCUCAG CUGCCACCGC U G 620forw GGACCGACAG 538 1082forw GCCCACCUUU 593 CGACAACUCC AGGGUGAUAG A C 1239rev CAGAAUCUUG 539 456forw GUUGCCUGGA 594 UCUCGGCUCA GCCGUUCCUG G C 1229rev UCUCGGCUCA 540 1391rev AAAAAGCGAU 595 GUGGGAUGCG CUUAGAUCAC U C 1584forw CCCCCCAACC 541 414forw GCACCUCCAA 596 AGCCCGCCCG AGUCGGCCAA A A 481rev GGUUGUAAAG 542 1182forw CUAUUACCUA 597 UUUUUUACGG AGUAGGUCCC A C 1625rev AAGGGAGCGG 543 192forw GGCCGCAGGA 598 CCGCCGACCG AGAGGAACGG C A 1896forw CGCCCCUCCC 544 875forw UCCCAGCCGG 599 GGGGACCUGC CAGUCUCCCA G C 1661rev CGCAACCCGG 545 1830forw GGUGGUGCAG 600 UGCGCUGCCG GGCCGUGAAG G C 383forw UGAGAUAAUG 546 214rev GGGUGCACGU 601 UGGGAUGCUA CCCACAGCUC A A 472forw AAGUGCAAUA 547 1724rev CAUGCACUUG 602 GUGCUAAAAA UCUGUAGUUC C A 614rev UAUUCAGUUA 548 1070forw GUGUAUAUAA 603 GAUAAACUCC AGGCCCACCU G U 1376rev UCACCUCGAC 549 337rev CUGGCCCAGU 604 UACCUUAAAA CAGUCAGGUU A U 166forw CCAUAAGGAA 550 850rev UUGUGGGAGA 605 ACUAUGUUAU CUGCCGGCUG G G 409rev AUAGAGUAGA 551 984rev UGAUCCUAAG 606 UGCUAAAUGC GAAUUGAAGG U U 1170rev UCUCCAGCCU 552 1086rev UGACUUCUGA 607 CUCCUUGAGC CUUUCAAUCA A U 1122rev CUACAUUAUU 553 843rev AGACUGCCGG 608 AAUCUUAAGG CUGGGAGGGU A U 1372forw CUUAGGCCCU 554 1014forw UUCAAUUCCU 609 AAAAUCUUCC UAGGAUCAUC U U 268rev AAAUUCCUAU 555 1348forw UGAUUUUGCC 610 UGCUUAUAAU AAGAACUUGU C C 1836rev GCUGACAUUU 556 1703rev AGGAGUCCCC 611 UUUGUUUGCU GCCCUUGCAA C A 183forw UAUGAUGAAU 557 244rev AAAGCGAACU 612 GUGAUAGUUU GCAUGUGUGA G G 1260rev CCCAGGCAGC 558 945rev UACCUUGUCG 613 ACUGACUACA UGAUAAGUGG G G 1034rev GUCUUGAUGA 559 1847forw AAGCAAGAAU 614 GGUAAAAAGA GGCCACAGAC G U 1027forw AAAAAAUCGU 560 1043forw UUGCCAAGAG 615 UACAAUUUAU AGAGAGCAGC G C 287forw GUACUCAAGA 561 50rev GGGCCGACCG 616 UUAUAAGCAA CGGCCUCCAG U G lOlrev UUAAUUUCUC 562 131forw AAGAGUUGGG 617 UCCUUUGCAU CUCUGUCAGC A C 1201rev CCUACCCUGC 563 1217forw AGAUACAUUU 618 CCCCUUCUCC CUUAGCACUA U U 1284forw CUGCUGUAGU 564 1763rev AGAAGCAAAA 619 CAGUGCUGCC GUACCACUAG U A 429rev CACUUAGCAC 565 431forw CAAAAUGAAU 620 AGUACCUUAC GGGCAGUGAG A C 355forw ACUUAGAUAU 566 1322forw GCAUGGUUUU 621 GAAGGGGAAA GUGGAAAAGU G A 661rev UGCAGUUUUA 567 972rev AUUGAAGGUA 622 CAUAUAAAUG UGGAUUUGGG A A 1703forw GUUGCGGAGG 568 629forw UGAGAGAUCA 623 GUGGGCCUGG UUUAACAUUU G A 1332forw AAAAAUGUGA 569 1003forw AAAUCCAUAC 624 UGAUCAAAAC CUUCAAUUCC U U 1219forw GGCAUUCUAA 570 1244forw UAAGAAAUGU 625 GGAGAAGGGG AAAAAAACCU G C 372forw AAAGAAGGGU 571 836rev CGGCUGGGAG 626 UUGAGAUAAU GGUUGGGGGU G G 192forw UGUGAUAGUU 572 1462forw UCCCACUCUG 627 UGGAGAAUAA UCACCCAGAG A C 531forw GCAGAUGCUA 573 764rev UUAUUGGAAC 628 UGAAAGAAAA GCUAAGCUUG A U 708rev UAUGAGCCAC 574 829rev GAGGGUUGGG 629 CGCACCCGGC GGUGGGGGGU C G 741rev CGCUUCGGCC 575 1445rev CUGCACUCCA 630 UCCCAAAGUG GCUCUGGGUG C A 765forw CCAGCACUUU 576 755forw CUUGGCGAUG 631 GGGAGGCCGA ACCUUGAGCA A G 769forw CACUUUGGGA 577 1436forw UUUCUUCUCU 632 GGCCGAAGCG UUCUUUUGAG G A 820rev UUUUGUGUUU 578 1453rev UGGGGACACU 633 UUAGUAGAGA GCACUCCAGC C U 877rev UGUCUCAGCC 579 1544forw UUCUCUCAGC 634 UCCUGAGUAG CUCCCAAGUA C G 886rev CGAUUCUCGU 580 1570rev CUGAAAAUAC 635 GUCUCAGCCU AAAAAAAUCA C G 905forw CUACUCAGGA 581 1621rev GCGGGCGGAU 636 GGCUGAGACA CACCUGAGGU C C 918rev GCUCACUGCA 582 1638rev CACUUUGGGA 637 ACCUCUGCCU GGCAGAAGCG C G 962rev UGCUCUUUCG 583 1666forw CGCUUCUGCC 638 CCCAGGCUGG UCCCAAAGUG A C

In some aspects, a guide RNA molecule can comprise a part of any sequence recited in Table 3 or Table 4. In some aspects, a guide RNA molecule can comprise the first about 20 nulceotides of any sequence recited in Table 3. In some aspects, a guide RNA molecule can comprise the first about 21 nucleotides of any sequence recited in Table 4.

TABLE Guide RNA Sequences Seq Seq Seq ID Seq ID Name Sequence NO Name Sequence NO 1015rev UGUUCAUAAA 639 1934rev GAGAAGCCCC 903 UUUACUGACA GGGCCGACCG UGG CGG 1025forw AAAAAAAUCG 640 1937forw CGAACCCCGC 904 UUACAAUUUA CUGGAGGCCG UGG CGG 1028forw AAAAUCGUUA 641 1941forw CCCCGCCUGG 905 CAAUUUAUGG AGGCCGCGGU UGG CGG 1037rev UCUUGAUGAG 642 1944rev GGUGCCUCCG 906 GUAAAAAGAG GAGAAGCCCC GGG GGG 1038rev GUCUUGAUGA 643 1945rev GGGUGCCUCC 907 GGUAAAAAGA GGAGAAGCCC GGG CGG 1039rev UGUCUUGAUG 644 1946forw CCUGGAGGCC 908 AGGUAAAAAG GCGGUCGGCC AGG CGG 103rev AAUUUCUCUC 645 1947forw CUGGAGGCCG 909 CUUUGCAUAU CGGUCGGCCC UGG GGG 1049rev AGUAGUGCUG 646 1948forw UGGAGGCCGC 910 UGUCUUGAUG GGUCGGCCCG AGG GGG 1059rev AGGGGACCUA 647 1956rev GCGGUGGCAG 911 CUUAGGUAAU UGGGUGCCUC AGG CGG 1066rev CAAUUCCAGG 648 1957forw CGGUCGGCCC 912 GGACCUACUU GGGGCUUCUC AGG CGG 106forw ACGGAGCGAG 649 1960forw UCGGCCCGGG 913 UCCCCGCGCG GCUUCUCCGG CGG AGG 1073forw UUUUAACCUA 650 1965rev CAACUCUUCG 914 UUACCUAAGU CGGUGGCAGU AGG GGG 1077rev UAUCUGCUAG 651 1966rev CCAACUCUUC 915 ACAAUUCCAG GCGGUGGCAG GGG UGG 1078rev GUAUCUGCUA 652 1986forw CCACUGCCAC 916 GACAAUUCCA CGCGAAGAGU GGG UGG 1079rev UGUAUCUGCU 653 1987forw CACUGCCACC 917 AGACAAUUCC GCGAAGAGUU AGG GGG 1081forw UAUUACCUAA 654 199forw UUUGGAGAAU 918 GUAGGUCCCC AAAUUGAAUG UGG AGG 1098rev UCCCUUUUAU 655 1rev CAGAGCCCAA 919 UAGGAAAGAA CUCUUCGCGG AGG UGG 1107rev GACUGAAUCU 656 203forw GAGAAUAAAU 920 CCCUUUUAUU UGAAUGAGGA AGG AGG 1116forw CGCCUUUCUU 657 203rev CCAUUGCCGG 921 UCCUAAUAAA CGAGGGGUGA AGG CGG 1117forw GCCUUUCUUU 658 206rev AACUGAUCAC 922 CCUAAUAAAA CAAAUCUCCA GGG GGG 1129rev CUACUACAUU 659 207rev UAACUGAUCA 923 AUUAAUCUUA CCAAAUCUCC AGG AGG 1139rev CCAGCAACAG 660 209forw AAAUUGAAUG 924 UGGACUCUAG AGGAAGGCCC AGG UGG 1149rev GAGAACAUUA 661 209rev AAGCCCCCAU 925 CCAGCAACAG UGCCGGCGAG UGG GGG 114forw GCUAAAUAUC 662 210rev CAAGCCCCCA 926 CAAUAUGCAA UUGCCGGCGA AGG GGG 1159forw CCUCUAGAGU 663 211rev ACAAGCCCCC 927 CCACUGUUGC AUUGCCGGCG UGG AGG 1168rev GCCUCUCCUU 664 216rev GGUUCACAAG 928 GAGCAGAGGA CCCCCAUUGC UGG CGG 116rev GGUGCACGUC 665 217forw UGAGGAAGGC 929 CCACAGCUCA CCUGGAGAUU GGG UGG 1172rev UCCAGCCUCU 666 217forw GCGCAUCCGU 930 CCUUGAGCAG CACCCCUCGC AGG CGG 1178forw CUGGUAAUGU 667 223forw CCGUCACCCC 931 UCUCUAAAUA UCGCCGGCAA AGG UGG 117rev GGGUGCACGU 668 224forw CGUCACCCCU 932 CCCACAGCUC CGCCGGCAAU AGG GGG 1182forw UUAAAGCCAU 669 225forw GUCACCCCUC 933 CCUCUGCUCA GCCGGCAAUG AGG GGG 1187forw GCCAUCCUCU 670 226forw UCACCCCUCG 934 GCUCAAGGAG CCGGCAAUGG AGG GGG 1191forw UCCUCUGCUC 671 237rev GCCCAGUCAG 935 AAGGAGAGGC UCAGGUUUGG UGG GGG 1193rev UUCCACAAAA 672 238rev GGCCCAGUCA 936 CCAUGCUGAU GUCAGGUUUG AGG GGG 1197forw GCUCAAGGAG 673 239rev UGGCCCAGUC 937 AGGCUGGAGA AGUCAGGUUU AGG GGG 1203forw AAAUAUUUUU 674 240rev CUGGCCCAGU 938 CCUAUCAGCA CAGUCAGGUU UGG UGG 1207forw AGGCUGGAGA 675 243rev AAGACUUGGC 939 AGGCAUUCUA ACUUUAUAUG AGG UGG 1211forw UUCCUAUCAG 676 245rev GCACACUGGC 940 CAUGGUUUUG CCAGUCAGUC UGG AGG 1213forw GAGAAGGCAU 677 255forw AACCCCCAAA 941 UCUAAGGAGA CCUGACUGAC AGG UGG 1214forw AGAAGGCAUU 678 256forw ACCCCCAAAC 942 CUAAGGAGAA CUGACUGACU GGG GGG 1215forw GAAGGCAUUC 679 257rev AUAAUCUUGA 943 UAAGGAGAAG GUACAAGACU GGG UGG 1216forw AAGGCAUUCU 680 259rev CCUGCCAAUU 944 AAGGAGAAGG UGCAGCACAC GGG UGG 1220forw CAUUCUAAGG 681 275forw UGGGCCAGUG 945 AGAAGGGGGC UGCUGCAAAU AGG UGG 1221forw AUUCUAAGGA 682 279forw CCAGUGUGCU 946 GAAGGGGGCA GCAAAUUGGC GGG AGG 1221forw CAUGGUUUUG 683 287forw UACUCAAGAU 947 UGGAAAAGUA UAUAAGCAAU AGG AGG 1225forw UAAGGAGAAG 684 28rev CCUCGCCCCC 948 GGGGCAGGGU GAGAGACCCG AGG CGG 1232forw AAGGGGGCAG 685 290forw CAAAUUGGCA 949 GGUAGGAACU GGAGACGUGA CGG AGG 1234rev CAAGACUCUA 686 295rev UUCAUUUUGG 950 GACAAGUUCU CCGACUUUGG UGG AGG 1241rev GAAUCUUGUC 687 298rev CCAUUCAUUU 951 UCGGCUCAGU UGGCCGACUU GGG UGG 1242rev AGAAUCUUGU 688 305forw CGUGAAGGCA 952 CUCGGCUCAG CCUCCAAAGU UGG CGG 1250rev ACUACAGCAG 689 308rev GGCUCACUGC 953 AAUCUUGUCU CCAUUCAUUU CGG UGG 1257forw AGAACUUGUC 690 318forw CCAAAGUCGG 954 UAGAGUCUUG CCAAAAUGAA AGG UGG 126forw CGGCGCGAUU 691 319forw CAAAGUCGGC 955 CCCUGAGCUG CAAAAUGAAU UGG GGG 127forw GGCGCGAUUC 692 31forw GAGUUGGGCU 956 CCUGAGCUGU CUGUCAGCCG GGG CGG 1281rev CUUUGUGAAA 693 329rev GGAACGGCUC 957 AUAGAUUCCC CAGGCAACCC AGG CGG 1281rev AGAUCACCUU 694 32forw AGUUGGGCUC 958 GAGUAAACUG UGUCAGCCGC AGG GGG 1283forw CUGCUGUAGU 695 330forw AAAAUGAAUG 959 CAGUGCUGCC GGCAGUGAGC UGG CGG 1284forw UGCUGUAGUC 696 331forw AAAUGAAUGG 960 AGUGCUGCCU GCAGUGAGCC GGG GGG 1295forw AGUAAGCCUC 697 331rev UUUCCCCUUC 961 AGUUUACUCA AUAUCUAAGU AGG AGG 1308rev GUUUUGAUCA 698 332forw AAUGAAUGGG 962 UCACAUUUUU CAGUGAGCCG UGG GGG 1332forw AAAAUGUGAU 699 338rev ACCCACGCAG 963 GAUCAAAACU GAACGGCUCC AGG AGG 1335forw UUCUUCUCUU 700 340forw GGCAGUGAGC 964 UCUUUUGAGA CGGGGUUGCC CGG UGG 1341rev CCAGCUCUGG 701 345rev CGGGAGAACC 965 GUGACAGAGU CACGCAGGAA GGG CGG 1342rev UCCAGCUCUG 702 346forw UAGUGCCUAC 966 GGUGACAGAG UUAGAUAUGA UGG AGG 1353rev GGACACUGCA 703 347forw AGUGCCUACU 967 CUCCAGCUCU UAGAUAUGAA GGG GGG 1354forw GAAUUAGUGU 704 348forw GUGCCUACUU 968 UCUGUGUCUU AGAUAUGAAG AGG GGG 1354rev GGGACACUGC 705 350rev GAAGACGGGA 969 ACUCCAGCUC GAACCCACGC UGG AGG 1357rev GAAUUCACAG 706 356forw UUAGAUAUGA 970 GAAGAUUUUA AGGGGAAAGA GGG AGG 1358rev GGAAUUCACA 707 356forw UGCCUGGAGC 971 GGAAGAUUUU CGUUCCUGCG AGG UGG 1361forw CCCACUCUGU 708 357forw UAGAUAUGAA 972 CACCCAGAGC GGGGAAAGAA UGG GGG 1369rev ACCUUAAAAA 709 357forw GCCUGGAGCC 973 UGGAAUUCAC GUUCCUGCGU AGG GGG 1374rev GGUUGCAGUG 710 364rev GGCAACAAAA 974 AGCCAAGAUG AGCGGAAGAC GGG GGG 1375rev AGGUUGCAGU 711 365rev AGGCAACAAA 975 GAGCCAAGAU AAGCGGAAGA GGG CGG 1376rev GAGGUUGCAG 712 372forw AAGAAGGGUU 976 UGAGCCAAGA UGAGAUAAUG UGG UGG 1379rev CACCUCGACU 713 372rev CCAUAAAAGG 977 ACCUUAAAAA CAACAAAAAG UGG CGG 137rev GCAUGUGUGA 714 373forw AGAAGGGUUU 978 GCCGAGUCCU GAGAUAAUGU GGG GGG 1382forw GGAGUGCAGU 715 385rev AGUUGUAAUA 979 GUCCCCAUCU CAACCAUAAA UGG AGG 1388forw UCCUGUGAAU 716 388forw AAUGUGGGAU 980 UCCAUUUUUA GCUAAGAGAA AGG UGG 138rev UGCAUGUGUG 717 391forw GUGGGAUGCU 981 AGCCGAGUCC AAGAGAAUGG UGG UGG 1395rev GCUAGAAACC 718 392forw CCGCUUUUUG 982 GAGGAGGCAG UUGCCUUUUA AGG UGG 1397forw UUCCAUUUUU 719 40forw UCUGUCAGCC 983 AAGGUAGUCG GCGGGUCUCU AGG CGG 1401rev AAAAUCGCUA 720 413rev CUCAACAAAA 984 GAAACCGAGG UCUGCAGAGC AGG AGG 1404rev UAUCCUCUGC 721 41forw CUGUCAGCCG 985 AGACCAGACG CGGGUCUCUC CGG GGG 1404rev GAGAAAAUCG 722 42forw UGUCAGCCGC 986 CUAGAAACCG GGGUCUCUCG AGG GGG 1407forw CACUGCAACC 723 434forw CUGCUCUGCA 987 UCUGCCUCCU GAUUUUGUUG CGG AGG 140forw GAAAUUAAAG 724 439forw UUUAGCAUCU 988 AUUUAAAAGC ACUCUAUGUA AGG AGG 140forw GAGCUGUGGG 725 43forw GUCAGCCGCG 989 ACGUGCACCC GGUCUCUCGG AGG GGG 1411forw UAGUCGAGGU 726 448rev GACUGGUCGA 990 GAACCGCGUC GAUCUACCUU UGG GGG 1421forw GAACCGCGUC 727 449rev GGACUGGUCG 991 UGGUCUGCAG AGAUCUACCU AGG UGG 1431rev UGUAAACCCA 728 452forw UGAGGUUUUU 992 GCUACUUGGG GCUUCUCCCA AGG AGG 1433forw GUCUGCAGAG 729 465rev CCACACCCCG 993 GAUAGAAAAA UUGAGGGGAC AGG UGG 1434rev GCCUGUAAAC 730 470rev UUCUCCCACA 994 CCAGCUACUU CCCCGUUGAG GGG GGG 1435rev UGCCUGUAAA 731 471rev GUUCUCCCAC 995 CCCAGCUACU ACCCCGUUGA UGG GGG 1436rev AACUAACUUG 732 472forw AGUGCAAUAG 996 AGGUAUCAGA UGCUAAAAAC GGG AGG 1437rev AAACUAACUU 733 472rev UGUUCUCCCA 997 GAGGUAUCAG CACCCCGUUG AGG AGG 1444forw CUCUCAGCCU 734 478forw AUCUCGACCA 998 CCCAAGUAGC GUCCCCUCAA UGG CGG 1445forw UCUCAGCCUC 735 479forw UCUCGACCAG 999 CCAAGUAGCU UCCCCUCAAC GGG GGG 1446rev UUAAAGGUGA 736 480forw CUCGACCAGU 1000 AACUAACUUG CCCCUCAACG AGG GGG 1453forw UCCCAAGUAG 737 485forw CCAGUCCCCU 1001 CUGGGUUUAC CAACGGGGUG AGG UGG 145rev AUCAUAACAU 738 486forw CAGUCCCCUC 1002 AGUUUCCUUA AACGGGGUGU UGG GGG 1462rev UUACUUCCGA 739 488rev CCAGGUUGUA 1003 CCUUCUUUAA AAGUUUUUUA AGG CGG 1462rev AAAAAAUCAG 740 48forw CCGCGGGUCU 1004 CCGGGUAUGG CUCGGGGGCG UGG AGG 1465rev ACAAAAAAAU 741 49forw CGCGGGUCUC 1005 CAGCCGGGUA UCGGGGGCGA UGG GGG 146forw UGGGACGUGC 742 4rev UGACAGAGCC 1006 ACCCAGGACU CAACUCUUCG CGG CGG 1470rev AAAAUACAAA 743 506rev UUUCUUUCAU 1007 AAAAUCAGCC AGCAUCUGCC GGG AGG 1471rev GAAAAUACAA 744 508forw CCGUAAAAAA 1008 AAAAAUCAGC CUUUACAACC CGG UGG 1472forw GUUAGUUUCA 745 530forw GCAGAUGCUA 1009 CCUUUAAAGA UGAAAGAAAA AGG AGG 1472forw CAGGCACACA 746 531forw CAGAUGCUAU 1010 CCACCAUACC GAAAGAAAAA CGG GGG 1476forw GUUUCACCUU 747 532forw AGAUGCUAUG 1011 UAAAGAAGGU AAAGAAAAAG CGG GGG 1495rev CCCUUCCGCA 748 536forw GCUAUGAAAG 1012 CGUCCGGGAA AAAAAGGGGA AGG UGG 1500rev CGUUGCCCUU 749 537forw CUAUGAAAGA 1013 CCGCACGUCC AAAAGGGGAU GGG GGG 1501rev ACGUUGCCCU 750 549forw AAGGGGAUGG 1014 UCCGCACGUC GAGAGAGAGA CGG AGG 1502forw UAAAGACGCA 751 54forw GUCUCUCGGG 1015 AAGCCUUUCC GGCGAGGGCG CGG AGG 1502forw UUUUGUAUUU 752 552forw GGGAUGGGAG 1016 UCAGUAAAGU AGAGAGAAGG UGG AGG 1503forw UUUGUAUUUU 753 553forw GGAUGGGAGA 1017 CAGUAAAGUU GAGAGAAGGA GGG GGG 1507forw UAUUUUCAGU 754 561forw UAGAAGAUCU 1018 AAAGUUGGGC AAAUGAACAU AGG UGG 150forw AUUUAAAAGC 755 563forw GAGAGAAGGA 1019 AGGAGCCAUA GGGAGAGAGA AGG UGG 1510forw CAAAGCCUUU 756 568forw AAGGAGGGAG 1020 CCCGGACGUG AGAGAUGGAG CGG AGG 1511forw UUCAGUAAAG 757 569forw AGGAGGGAGA 1021 UUGGGCAGGC GAGAUGGAGA UGG GGG 1514forw GCCUUUCCCG 758 574rev CAUAAACCGA 1022 GACGUGCGGA UGACCAUUAA AGG AGG 1514rev CACCUGAGGU 759 57forw AACAAGCGCU 1023 CAGGAGUUCG AUGACUAGCA AGG AGG 1515forw CCUUUCCCGG 760 581forw UGGAAAUUGU 1024 ACGUGCGGAA GUUCCUUUAA GGG UGG 1523rev CGGGCGGAUC 761 588forw UGUGUUCCUU 1025 ACCUGAGGUC UAAUGGUCAU AGG CGG 1524rev UCCAUUUCCG 762 597rev AAAAAGAAAC 1026 GCCAUGAGGA UUCUAACCUC AGG UGG 1528rev AAGUUCCAUU 763 598forw UUUACUUUUC 1027 UCCGGCCAUG UUUCAGAUCG AGG AGG 1528rev AGAAGCGGGC 764 601forw UGGUCAUCGG 1028 GGAUCACCUG UUUAUGCCAG AGG AGG 1532forw GGCCUCGAAC 765 602rev CUCCGUGGAG 1029 UCCUGACCUC UUGUCGCUGU AGG CGG 1533forw AAGGGCAACG 766 60forw CGGGGGCGAG 1030 UCCUUCCUCA GGCGAGGUUC UGG AGG 1536rev GGAAAUUAAA 767 617rev AUUCAGUUAG 1031 GUUCCAUUUC AUAAACUCCG CGG UGG 1537forw GCAACGUCCU 768 620forw GACCGACAGC 1032 UCCUCAUGGC GACAACUCCA CGG CGG 1539rev CUUUGGGAGG 769 632rev ACUGCUCAAG 1033 CAGAAGCGGG GUCAUCGCCA CGG AGG 1542rev GCACUUUGGG 770 635forw UUUUUUGAAA 1034 AGGCAGAAGC AAUUAGACCU GGG UGG 1543forw UCCUUCCUCA 771 63rev UCCUCUUCCU 1035 UGGCCGGAAA GCGGCCUGAA UGG AGG 1543rev AGCACUUUGG 772 644rev GGGUUAUAUC 1036 GAGGCAGAAG CUACUGCUCA CGG AGG 1552rev UGUAAUCCCA 773 655forw UGGCGAUGAC 1037 GCACUUUGGG CUUGAGCAGU AGG AGG 1555rev GCCUGUAAUC 774 663rev CAGUUUUACA 1038 CCAGCACUUU UAUAAAUGAC GGG AGG 1556rev CGCCUGUAAU 775 664rev UUGGAACGCU 1039 CCCAGCACUU AAGCUUGUGG UGG GGG 1557rev GCGGGCUGGU 776 665rev AUUGGAACGC 1040 UGGGGGGAAC UAAGCUUGUG GGG GGG 1558rev GGCGGGCUGG 777 666rev UAUUGGAACG 1041 UUGGGGGGAA CUAAGCUUGU CGG GGG 1563rev UCUCGGGCGG 778 667rev UUAUUGGAAC 1042 GCUGGUUGGG GCUAAGCUUG GGG UGG 1564rev CUCUCGGGCG 779 683rev UAUGCCUAGU 1043 GGCUGGUUGG GUUCCGUUAU GGG UGG 1565forw GCUUCUGCCU 780 68forw UGACUAGCAA 1044 CCCAAAGUGC GGUUAAGUGA UGG AGG 1565rev UCUCUCGGGC 781 690forw CAAGCUUAGC 1045 GGGCUGGUUG GUUCCAAUAA GGG CGG 1566forw CUUCUGCCUC 782 694forw UAUGUAAAAC 1046 CCAAAGUGCU UGCACUAUAC GGG UGG 1566rev CUCUCUCGGG 783 697rev CCGGCCGCGA 1047 CGGGCUGGUU AUUUUUAUAA GGG UGG 1567rev ACUCUCUCGG 784 699forw CGUUCCAAUA 1048 GCGGGCUGGU ACGGAACACU UGG AGG 1571rev AGUCACUCUC 785 69forw GGGCGAGGUU 1049 UCGGGCGGGC CAGGCCUUUC UGG AGG 1574forw UCCCAAAGUG 786 713forw CUGGCCAUUA 1050 CUGGGAUUAC UAAAAAUUCG AGG CGG 1575rev UGAGAGUCAC 787 714forw ACACUAGGCA 1051 UCUCUCGGGC UAAUGAAAGA GGG CGG 1576rev GUGAGAGUCA 788 716rev CAGGUAUGAG 1052 CUCUCUCGGG CCACCGCACC CGG CGG 1579rev CUCGUGAGAG 789 717forw CCAUUAUAAA 1053 UCACUCUCUC AAUUCGCGGC GGG CGG 1580rev UCUCGUGAGA 790 718forw CAUUAUAAAA 1054 GUCACUCUCU AUUCGCGGCC CGG GGG 1583rev GGAUCUUAGU 791 71rev ACUUUAAGCC 1055 CCCCGCACGG UUUCAGUCCC UGG UGG 1586rev AAGGGAUCUU 792 723forw UAAAAAUUCG 1056 AGUCCCCGCA CGGCCGGGUG CGG CGG 1591forw UACAGGCGUG 793 726forw AAAUUCGCGG 1057 AGCCACCGUG CCGGGUGCGG CGG UGG 1592forw ACAGGCGUGA 794 72rev UCGCUCCGUU 1058 GCCACCGUGC CCUCUUCCUG GGG CGG 1593forw CAGGCGUGAG 795 731rev AGGGUUGGGG 1059 CCACCGUGCG GUGGGGGGUG GGG UGG 1604rev AUUGGCCAAG 796 735rev UCCCAAAGUG 1060 CUGACUCUCG CUGGGAUUAC CGG AGG 1604rev GAGUCCCCGC 797 736rev CUGGGAGGGU 1061 CCUUGCAAAA UGGGGGUGGG GGG GGG 1605rev GGAGUCCCCG 798 737rev GCUGGGAGGG 1062 CCCUUGCAAA UUGGGGGUGG AGG GGG 1614forw GGACUAAGAU 799 738rev GGCUGGGAGG 1063 CCCUUUUGCA GUUGGGGGUG AGG GGG 1615forw GACUAAGAUC 800 739rev CGGCUGGGAG 1064 CCUUUUGCAA GGUUGGGGGU GGG GGG 1618forw UAAGAUCCCU 801 73forw AGCAAGGUUA 1065 UUUGCAAGGG AGUGAAGGCC CGG AGG 1619forw GAGAGCCGCG 802 740rev CCGGCUGGGA 1066 AGAGUCAGCU GGGUUGGGGG UGG UGG 1619forw AAGAUCCCUU 803 743rev CUUCGGCCUC 1067 UUGCAAGGGC CCAAAGUGCU GGG GGG 1620forw AGAUCCCUUU 804 743rev CUGCCGGCUG 1068 UGCAAGGGCG GGAGGGUUGG GGG GGG 1622rev CGGCCGCCGA 805 744rev GCUUCGGCCU 1069 CCGCACGGAU CCCAAAGUGC UGG UGG 1626rev AUGCACUUGU 806 744rev ACUGCCGGCU 1070 CUGUAGUUCA GGGAGGGUUG AGG GGG 1627rev GGGAGCGGCC 807 745rev GACUGCCGGC 1071 GCCGACCGCA UGGGAGGGUU CGG GGG 1632forw GUCAGCUUGG 808 746rev AGACUGCCGG 1072 CCAAUCCGUG CUGGGAGGGU CGG UGG 1636forw GCUUGGCCAA 809 74forw GCAAGGUUAA 1073 UCCGUGCGGU GUGAAGGCCA CGG GGG 1639forw UGGCCAAUCC 810 750rev UGGGAGACUG 1074 GUGCGGUCGG CCGGCUGGGA CGG GGG 1642rev GAGUCGGCUU 811 751rev GUGGGAGACU 1075 AUAAAGGGAG GCCGGCUGGG CGG AGG 1647rev CGGGCGAGUC 812 753forw UACCUGUAAU 1076 GGCUUAUAAA CCCAGCACUU GGG UGG 1648rev CCGGGCGAGU 813 754forw ACCUGUAAUC 1077 CGGCUUAUAA CCAGCACUUU AGG GGG 1658rev CGGUGCGCUG 814 754rev CUUGUGGGAG 1078 CCGGGCGAGU ACUGCCGGCU CGG GGG 1665rev GAAGCAAAAG 815 755rev UCUUGUGGGA 1079 UACCACUAGA GACUGCCGGC UGG UGG 1666rev CCGCAACCCG 816 757forw UGUAAUCCCA 1080 GUGCGCUGCC GCACUUUGGG GGG AGG 1667rev UCCGCAACCC 817 759rev CAAUUCUUGU 1081 GGUGCGCUGC GGGAGACUGC CGG CGG 1668forw CCUUUAUAAG 818 760forw CCACCCCCAA 1082 CCGACUCGCC CCCUCCCAGC CGG CGG 1673forw UUUGUUCUUA 819 760rev CUCAAGUGAU 1083 CUCCAUCUAG CCACCCGCUU UGG CGG 1678rev CAGGCCCACC 820 766forw AGCACUUUGG 1084 CUCCGCAACC GAGGCCGAAG CGG CGG 1679forw CGACUCGCCC 821 767forw GCACUUUGGG 1085 GGCAGCGCAC AGGCCGAAGC CGG GGG 1680forw GACUCGCCCG 822 769rev AAAUCAGAGC 1086 GCAGCGCACC CAAUUCUUGU GGG GGG 1686forw CCCGGCAGCG 823 76forw GUUCAGGCCU 1087 CACCGGGUUG UUCAGGCCGC CGG AGG 1689forw GGCAGCGCAC 824 770forw CUUUGGGAGG 1088 CGGGUUGCGG CCGAAGCGGG AGG UGG 1689rev CACCACAAAU 825 770rev GAAAUCAGAG 1089 GUUGUAAAUG CCAAUUCUUG UGG UGG 1690forw GCAGCGCACC 826 780forw CGGCAGUCUC 1090 GGGUUGCGGA CCACAAGAAU GGG UGG 1693forw GCGCACCGGG 827 783rev CAGGCUGGUC 1091 UUGCGGAGGG UCGAACGCCA UGG GGG 1694forw CGCACCGGGU 828 784rev CCAGGCUGGU 1092 UGCGGAGGGU CUCGAACGCC GGG AGG 1697rev AAAUGGCCAC 829 786forw CGGGUGGAUC 1093 CACCCCUCCC ACUUGAGCCC AGG UGG 1699forw CGGGUUGCGG 830 792rev CCAUUAGCUU 1094 AGGGUGGGCC AUUUUCUUAA UGG AGG 1700forw GGGUUGCGGA 831 798rev UUUCACCAUG 1095 GGGUGGGCCU UUGCCCAGGC GGG UGG 1703forw UUGCGGAGGG 832 802rev GGGGUUUCAC 1096 UGGGCCUGGG CAUGUUGCCC AGG AGG 1704forw UGCGGAGGGU 833 804forw CCUGGCGUUC 1097 GGGCCUGGGA GAGACCAGCC GGG UGG 1705forw GCGGAGGGUG 834 805forw CUGGCGUUCG 1098 GGCCUGGGAG AGACCAGCCU GGG GGG 1707forw CUCCACAUUU 835 812forw CCUUUAAGAA 1099 ACAACAUUUG AAUAAGCUAA UGG UGG 1708forw GAGGGUGGGC 836 813forw CGAGACCAGC 1100 CUGGGAGGGG CUGGGCAACA UGG UGG 170rev UCGGCGUUCC 837 815rev UUUGUUUCUU 1101 CCCCACCAAC UCAACCUAGU AGG GGG 1710forw CACAUUUACA 838 816rev GUUUGUUUCU 1102 ACAUUUGUGG UUCAACCUAG UGG UGG 1711forw GGUGGGCCUG 839 821forw AAAUAAGCUA 1103 GGAGGGGUGG AUGGCCCACU UGG AGG 1714rev AGUUAGGGUU 840 821rev UGUGUUUUUA 1104 AGACAAAAAA GUAGAGACGG UGG GGG 1716forw UACAACAUUU 841 822rev UUGUGUUUUU 1105 GUGGUGGUGC AGUAGAGACG AGG GGG 1717forw ACAACAUUUG 842 823rev UUUGUGUUUU 1106 UGGUGGUGCA UAGUAGAGAC GGG GGG 1720rev CUGUGGCCAU 843 824rev UUUUGUGUUU 1107 UCUUGCUUCA UUAGUAGAGA CGG CGG 1729rev GCCUACGCCC 844 82forw GCCUUUCAGG 1108 UUCUCAGUUA CCGCAGGAAG GGG AGG 1730rev CGCCUACGCC 845 839forw CUAGGUUGAA 1109 CUUCUCAGUU AGAAACAAAC AGG AGG 1734forw GCAGGGCCGU 846 83forw AGUGAAGGCC 1110 GAAGCAAGAA AGGGACUGAA UGG AGG 1737rev AGAAAAACAU 847 842rev GUCGUGAUAA 1111 UCCCAGUCUG GUGGGCAGAA UGG UGG 1741forw UUGUCUAACC 848 850rev AUUACCUUGU 1112 CUAACUGAGA CGUGAUAAGU AGG GGG 1742forw UGUCUAACCC 849 851rev AAUUACCUUG 1113 UAACUGAGAA UCGUGAUAAG GGG UGG 1745forw AAGCAAGAAU 850 853forw CUAAAAACAC 1114 GGCCACAGAC AAAAACUAGC UGG UGG 1746forw AGCAAGAAUG 851 854forw UAAAAACACA 1115 GCCACAGACU AAAACUAGCU GGG GGG 1748forw ACCCUAACUG 852 859forw ACACAAAAAC 1116 AGAAGGGCGU UAGCUGGGCG AGG UGG 1753rev GCGCGCGGGG 853 862forw CAAAAACUAG 1117 AGCAAAAGCA CUGGGCGUGG CGG UGG 175forw CAUGCAGUUC 854 866forw AACUAGCUGG 1118 GCUUUCCUGU GCGUGGUGGC UGG AGG 1766rev AGCGAGAAAA 855 866forw UCUGCCCACU 1119 ACAGCGCGCG UAUCACGACA GGG AGG 1767rev CAGCGAGAAA 856 871rev UCCUGAGUAG 1120 AACAGCGCGC CUGGGAUUAC GGG AGG 1768rev UCAGCGAGAA 857 874rev UUGAAGGUAU 1121 AAACAGCGCG GGAUUUGGGA CGG CGG 178forw GCAGUUCGCU 858 878rev GGAAUUGAAG 1122 UUCCUGUUGG GUAUGGAUUU UGG GGG 1798forw UUUUUCUCGC 859 879rev UCUCAGCCUC 1123 UGACUUUCAG CUGAGUAGCU CGG GGG 1799forw UUUUCUCGCU 860 879rev AGGAAUUGAA 1124 GACUUUCAGC GGUAUGGAUU GGG UGG 179forw CAGUUCGCUU 861 87forw UCAGGCCGCA 1125 UCCUGUUGGU GGAAGAGGAA GGG CGG 1802forw UCUCGCUGAC 862 880rev GUCUCAGCCU 1126 UUUCAGCGGG CCUGAGUAGC CGG UGG 180forw AGUUCGCUUU 863 885rev AUCCUAAGGA 1127 CCUGUUGGUG AUUGAAGGUA GGG UGG 1810rev CGGUGGAAGG 864 890forw GCCUGUAAUC 1128 CGGCAGGCCG CCAGCUACUC AGG AGG 1813forw UUCAGCGGGC 865 890rev AGAUGAUCCU 1129 GGAAAAGCCU AAGGAAUUGA CGG AGG 1816rev AAUGAACGGU 866 893forw UGUAAUCCCA 1130 GGAAGGCGGC GCUACUCAGG AGG AGG 181forw UUAUGAUGAA 867 899rev ACUACCCCCA 1131 UGUGAUAGUU GAUGAUCCUA UGG AGG 181forw GUUCGCUUUC 868 903forw AUCCAUACCU 1132 CUGUUGGUGG UCAAUUCCUU GGG AGG 1820rev CUAGAAUGAA 869 912forw UUCAAUUCCU 1133 CGGUGGAAGG UAGGAUCAUC CGG UGG 1823rev GCUCUAGAAU 870 913forw UCAAUUCCUU 1134 GAACGGUGGA AGGAUCAUCU AGG GGG 1827rev GUUUGCUCUA 871 914forw CAAUUCCUUA 1135 GAAUGAACGG GGAUCAUCUG UGG GGG 182forw UUCGCUUUCC 872 915forw AAUUCCUUAG 1136 UGUUGGUGGG GAUCAUCUGG GGG GGG 1830rev UUUGUUUGCU 873 919rev CACUGCAACC 1137 CUAGAAUGAA UCUGCCUCCC CGG GGG 1866forw AAACAAAAAA 874 920rev UCACUGCAAC 1138 UGUCAGCUGC CUCUGCCUCC UGG CGG 1869rev GGUCCCCGGG 875 921forw ACACGAGAAU 1139 AGGGGCGAAC CGCUUGAACC GGG CGG 1870rev AGGUCCCCGG 876 922forw CACGAGAAUC 1140 GAGGGGCGAA GCUUGAACCC CGG GGG 1877rev CCGCCGCAGG 877 924rev CCCCUGGCUG 1141 UCCCCGGGAG CUCUCUCUCU GGG UGG 1878rev CCCGCCGCAG 878 925forw GAGAAUCGCU 1142 GUCCCCGGGA UGAACCCGGG GGG AGG 1879rev ACCCGCCGCA 879 931forw CGCUUGAACC 1143 GGUCCCCGGG CGGGAGGCAG AGG AGG 1882rev GCGACCCGCC 880 940rev GGCCUUUAUA 1144 GCAGGUCCCC UACACACCCC GGG UGG 1883rev GGCGACCCGC 881 942forw UGCCAAGAGA 1145 CGCAGGUCCC GAGAGCAGCC CGG AGG 1884forw GCUGGCCCGU 882 943forw GCCAAGAGAG 1146 UCGCCCCUCC AGAGCAGCCA CGG GGG 1885forw CUGGCCCGUU 883 944forw CCAAGAGAGA 1147 CGCCCCUCCC GAGCAGCCAG GGG GGG 1886forw UGGCCCGUUC 884 944rev GGAGUCUAGU 1148 GCCCCUCCCG GGCGUGAUCU GGG CGG 1890rev CUGGGCAGGC 885 955rev CCAGGCUGGA 1149 GACCCGCCGC UGGAGUCUAG AGG UGG 1894forw UCGCCCCUCC 886 958forw AGCCAGGGGU 1150 CGGGGACCUG GUGUAUAUAA CGG AGG 1897forw CCCCUCCCGG 887 961rev CAGGCUAUCA 1151 GGACCUGCGG CCCUAAAGGU CGG GGG 1898forw CCCUCCCGGG 888 962rev UCAGGCUAUC 1152 GACCUGCGGC ACCCUAAAGG GGG UGG 189rev GGGUGACGGA 889 965rev GCUCUUUCGC 1153 UGCGCACGAU CCAGGCUGGA CGG UGG 1904rev GCGGGGUUCG 890 965rev GAUUCAGGCU 1154 GGGGCUGGGC AUCACCCUAA AGG AGG 1908rev CCAGGCGGGG 891 969rev UCUUGCUCUU 1155 UUCGGGGGCU UCGCCCAGGC GGG UGG 1909rev UCCAGGCGGG 892 970forw GUAUAUAAAG 1156 GUUCGGGGGC GCCCACCUUU UGG AGG 1913rev GGCCUCCAGG 893 971forw UAUAUAAAGG 1157 CGGGGUUCGG CCCACCUUUA GGG GGG 1914rev CGGCCUCCAG 894 973rev GGAGUCUUGC 1158 GCGGGGUUCG UCUUUCGCCC GGG AGG 1915rev GCGGCCUCCA 895 975forw CCACUAGACU 1159 GGCGGGGUUC CCAUCCAGCC GGG UGG 1916rev CGCGGCCUCC 896 976forw CACUAGACUC 1160 AGGCGGGGUU CAUCCAGCCU CGG GGG 1921rev CCGACCGCGG 897 97rev AGGGAAUCGC 1161 CCUCCAGGCG GCCGCGCGCG GGG GGG 1922rev GCCGACCGCG 898 980rev ACUUUCAAUC 1162 GCCUCCAGGC AUCAGGAUUC GGG AGG 1923rev GGCCGACCGC 899 987rev ACUUCUGACU 1163 GGCCUCCAGG UUCAAUCAUC CGG AGG 1926rev CCGGGCCGAC 900 98rev CAGGGAAUCG 1164 CGCGGCCUCC CGCCGCGCGC AGG GGG 1928forw CCCAGCCCCC 901 994rev AACGAUUUUU 1165 GAACCCCGCC UUUUUUGAGA UGG CGG 1931forw AGCCCCCGAA 902 99rev UCAGGGAAUC 1166 CCCCGCCUGG GCGCCGCGCG AGG CGG

TABLE 4 Guide RNA sequences Seq Seq Seq ID Seq Sequence ID Name Sequence NO Name NO 1420forw GUGAACCGCGU 1167 967rev AGUCUUGCUCU 1222 CUGGUCUGC UUCGCCCAGG AGAGGAU CUGGAU 1591rev GCUGACUCUCG 1168 991rev UAACGAUUUUU 1223 CGGCUCUCG UUUUUUGAG UGAGAGU ACGGAGU 634forw AACUCCACGGA 1169 94rev GCCCAACUCUU 1224 GUUUAUCUA CGCGGUGGCA ACUGAAU GUGGGU 640forw ACGGAGUUUAU 1170 302rev CCCCAUUGCCG 1225 CUAACUGAA GCGAGGGGUG UACGAGU ACGGAU 1688forw CCGGCAGCGCA 1171 997rev AACUACCCCCA 1226 CCGGGUUGC GAUGAUCCUA GGAGGGU AGGAAU 716forw GGCCAUUAUAA 1172 237rev ACUGCAUGUGU 1227 AAAUUCGCG GAGCCGAGUC GCCGGGU CUGGGU 1608forw AGUGACUCUCA 1173 309rev CACAAGCCCCC 1228 CGAGAGCCG AUUGCCGGCG CGAGAGU AGGGGU 1678forw GCCGACUCGCC 1174 579forw GAUCUCGACCA 1229 CGGCAGCGC GUCCCCUCAA ACCGGGU CGGGGU 1728rev GGCGCCUACGC 1175 llOforw GAGGCACCCAC 1230 CCUUCUCAG UGCCACCGCG UUAGGGU AAGAGU 620forw GGACCGACAGC 1176 1082forw GCCCACCUUUA 1231 GACAACUCC GGGUGAUAGC ACGGAGU CUGAAU 1239rev CAGAAUCUUGU 1177 456forw GUUGCCUGGAG 1232 CUCGGCUCA CCGUUCCUGC GUGGGAU GUGGGU 1229rev UCUCGGCUCAG 1178 1391rev AAAAAGCGAUC 1233 UGGGAUGCG UUAGAUCACC UCCGAGU UUGAGU 1584forw CCCCCCAACCA 1179 414forw GCACCUCCAAA 1234 GCCCGCCCG GUCGGCCAAA AGAGAGU AUGAAU 481rev GGUUGUAAAGU 1180 1182forw CUAUUACCUAA 1235 UUUUUACG GUAGGUCCCC GACAGAAU UGGAAU 1625rev AAGGGAGCGGC 1181 192forw GGCCGCAGGAA 1236 CGCCGACCG GAGGAACGG CACGGAU AGCGAGU 1896forw CGCCCCUCCCG 1182 875forw UCCCAGCCGGC 1237 GGGACCUGC AGUCUCCCAC GGCGGGU AAGAAU 1661rev CGCAACCCGGU 1183 1830forw GGUGGUGCAGG 1238 GCGCUGCCG GCCGUGAAG GGCGAGU CAAGAAU 383forw UGAGAUAAUGU 1184 214rev GGGUGCACGUC 1239 GGGAUGCU CCACAGCUCA AAGAGAAU GGGAAU 472forw AAGUGCAAUAG 1185 1724rev CAUGCACUUGU 1240 UGCUAAAA CUGUAGUUCA ACAGGAGU AGGAGU 614rev UAUUCAGUUAG 1186 1070forw GUGUAUAUAAA 1241 AUAAACUCC GGCCCACCUU GUGGAGU UAGGGU 1376rev UCACCUCGACU 1187 337rev CUGGCCCAGUC 1242 ACCUUAAAA AGUCAGGUUU AUGGAAU GGGGGU 166forw CCAUAAGGAAA 1188 850rev UUGUGGGAGAC 1243 CUAUGUUAU UGCCGGCUG GAUGAAU GGAGGGU 409rev AUAGAGUAGAU 1189 984rev UGAUCCUAAGG 1244 GCUAAAUG AAUUGAAGG CUUUGAGU UAUGGAU 1170rev UCUCCAGCCUC 1190 1086rev UGACUUCUGAC 1245 UCCUUGAGC UUUCAAUCAU AGAGGAU CAGGAU 1122rev CUACAUUAUUA 1191 843rev AGACUGCCGGC 1246 AUCUUAAGG UGGGAGGGU ACUGAAU UGGGGGU 1372forw CUUAGGCCCUA 1192 1014forw UUCAAUUCCUU 1247 AAAUCUUCC AGGAUCAUCU UGUGAAU GGGGGU 268rev AAAUUCCUAUU 1193 1348forw UGAUUUUGCCA 1248 GCUUAUAAU AGAACUUGU CUUGAGU CUAGAGU 1836rev GCUGACAUUUU 1194 1703rev AGGAGUCCCCG 1249 UUGUUUGCU CCCUUGCAAA CUAGAAU AGGGAU 183forw UAUGAUGAAUG 1195 244rev AAAGCGAACUG 1250 UGAUAGUU CAUGUGUGA UGGAGAAU GCCGAGU 1260rev CCCAGGCAGCA 1196 945rev UACCUUGUCGU 1251 CUGACUACA GAUAAGUGG GCAGAAU GCAGAAU 1034rev GUCUUGAUGAG 1197 1847forw AAGCAAGAAUG 1252 GUAAAAAG GCCACAGACU AGGGGAGU GGGAAU 1027forw AAAAAAUCGUU 1198 1043forw UUGCCAAGAGA 1253 ACAAUUUA GAGAGCAGCC UGGUGGAU AGGGGU 287forw GUACUCAAGAU 1199 50rev GGGCCGACCGC 1254 UAUAAGCAA GGCCUCCAGG UAGGAAU CGGGGU lOlrev UUAAUUUCUCU 1200 131forw AAGAGUUGGGC 1255 CCUUUGCAU UCUGUCAGCC AUUGGAU GCGGGU 1201rev CCUACCCUGCC 1201 1217forw AGAUACAUUUC 1256 CCCUUCUCC UUAGCACUA UUAGAAU UUAGAAU 1284forw CUGCUGUAGUC 1202 1763rev AGAAGCAAAAG 1257 AGUGCUGCC UACCACUAG UGGGAAU AUGGAGU 429rev CACUUAGCACA 1203 431forw CAAAAUGAAUG 1258 GUACCUUAC GGCAGUGAG AUAGAGU CCGGGGU 355forw ACUUAGAUAUG 1204 1322forw GCAUGGUUUUG 1259 AAGGGGAA UGGAAAAGU AGAAGGGU AAGGAAU 661rev UGCAGUUUUAC 1205 972rev AUUGAAGGUAU 1260 AUAUAAAU GGAUUUGGG GACAGGAU ACGGAAU 1703forw GUUGCGGAGGG 1206 629forw UGAGAGAUCAU 1261 UGGGCCUGG UUAACAUUU GAGGGGU AAUGAAU 1332forw AAAAAUGUGAU 1207 1003forw AAAUCCAUACC 1262 GAUCAAAA UUCAAUUCCU CUAGGAAU UAGGAU 1219forw GGCAUUCUAAG 1208 1244forw UAAGAAAUGUA 1263 GAGAAGGG AAAAAACCU GGCAGGGU CUAGAGU 372forw AAAGAAGGGUU 1209 836rev CGGCUGGGAGG 1264 UGAGAUAA GUUGGGGGU UGUGGGAU GGGGGGU 192forw UGUGAUAGUUU 1210 1462forw UCCCACUCUGU 1265 GGAGAAUA CACCCAGAGC AAUUGAAU UGGAGU 531forw GCAGAUGCUAU 1211 764rev UUAUUGGAACG 1266 GAAAGAAA CUAAGCUUG AAGGGGAU UGGGGGU 708rev UAUGAGCCACC 1212 829rev GAGGGUUGGGG 1267 GCACCCGGC GUGGGGGGU CGCGAAU GUGGAAU 741rev CGCUUCGGCCU 1213 1445rev CUGCACUCCAG 1268 CCCAAAGUG CUCUGGGUGA CUGGGAU CAGAGU 765forw CCAGCACUUUG 1214 755forw CUUGGCGAUGA 1269 GGAGGCCGA CCUUGAGCAG AGCGGGU UAGGAU 769forw CACUUUGGGAG 1215 1436forw UUUCUUCUCUU 1270 GCCGAAGCG UCUUUUGAG GGUGGAU ACGGAGU 820rev UUUUGUGUUUU 1216 1453rev UGGGGACACUG 1271 UAGUAGAG CACUCCAGCU ACGGGGGU CUGGGU 877rev UGUCUCAGCCU 1217 1544forw UUCUCUCAGCC 1272 CCUGAGUAG UCCCAAGUAG CUGGGAU CUGGGU 886rev CGAUUCUCGUG 1218 1570rev CUGAAAAUACA 1273 UCUCAGCCU AAAAAAUCA CCUGAGU GCCGGGU 905forw CUACUCAGGAG 1219 1621rev GCGGGCGGAUC 1274 GCUGAGACA ACCUGAGGUC CGAGAAU AGGAGU 918rev GCUCACUGCAA 1220 1638rev CACUUUGGGAG 1275 CCUCUGCCU GCAGAAGCG CCCGGGU GGCGGAU 962rev UGCUCUUUCGC 1221 1666forw CGCUUCUGCCU 1276 CCAGGCUGG CCCAAAGUGC AUGGAGU UGGGAU

In some aspects, a guide RNA molecule can be chemically synthesized using methods standard in the art. In some aspects, a guide RNA molecule can be chemically synthesized such that the guide RNA molecule comprises at least one chemical modification. In some aspects, a guide RNA molecule can be produced by in vitro transcription methods standard in the art, including, but not limited to, in vitro transcription using a plasmid template, in vitro transcription using a PCR-based template. In some aspects, in vitro transcription methods can be performed such that the produced guide RNA molecules comprise at least one chemical modification.

In some aspects, any of the compositions of the present disclosure can further comprise at least one mRNA and/or polynucleotide encoding a fusion protein comprising at least a portion of the MS2 coat protein (MCP) and at least one transactivation molecule. In some aspects, the at least one polynucleotide can be a plasmid comprising a nucleic acid encoding a fusion protein comprising at least a portion of the MS2 coat protein (MCP) and at least one transactivation molecule operably linked to at least one promoter sufficient to drive expression of the fusion protein.

In some aspects, any of the compositions of the present disclosure can further comprise at least one mRNA and/or polynucleotide encoding a fusion protein comprising at least a portion of MS2 coat protein (MCP) and at least one VP64 transactivation molecule. In some aspects, the at least one polynucleotide can be a plasmid comprising a nucleic acid encoding a fusion protein comprising at least a portion of MS2 coat protein (MCP) and at least one VP64 transactivation molecule operably linked to at least one promoter sufficient to drive expression of the fusion protein.

In some aspects, any of the compositions of the present disclosure can further comprise at least one mRNA and/or polynucleotide encoding a fusion protein comprising at least a portion of MS2 coat protein (MCP) and at least one P65-HSF transactivation molecule. In some aspects, the at least one polynucleotide can be a plasmid comprising a nucleic acid encoding a fusion protein comprising at least a portion of MS2 coat protein (MCP) and at least one P65-HSF transactivation molecule operably linked to at least one promoter sufficient to drive expression of the fusion protein.

In some aspects, any of the compositions of the present disclosure can further comprise at least one mRNA and/or polynucleotide encoding a fusion protein comprising at least one antibody that binds to the SunTag peptide and at least one transactivation molecule. In some aspects, the at least one polynucleotide can be a plasmid comprising a nucleic acid encoding a fusion protein comprising at least one antibody that binds to the SunTag peptide and at least one transactivation molecule operably linked to at least one promoter sufficient to drive expression of the fusion protein.

In some aspects, any of the compositions of the present disclosure can further comprise at least one mRNA and/or polynucleotide encoding a fusion protein comprising at least one antibody that binds to the SunTag peptide and at least one P65-HSF transactivation molecule. In some aspects, the at least one polynucleotide can be a plasmid comprising a nucleic acid encoding a fusion protein comprising at least one antibody that binds to the SunTag peptide and at least one P65-HSF transactivation molecule operably linked to at least one promoter sufficient to drive expression of the fusion protein.

In some aspects, any of the compositions of the present disclosure can further comprise at least one mRNA and/or polynucleotide encoding a fusion protein comprising at least one antibody that binds to the SunTag peptide and at least one VP64 transactivation molecule. In some aspects, the at least one polynucleotide can be a plasmid comprising a nucleic acid encoding a fusion protein comprising at least one antibody that binds to the SunTag peptide and at least one VP64 transactivation molecule operably linked to at least one promoter sufficient to drive expression of the fusion protein.

In some aspects, any composition of the present disclosure can further comprise at least one mRNA and/or polynucleotide encoding at least one rejuvenating factor. In some aspects, the at least one polynucleotide can be a plasmid comprising a nucleic acid encoding at least one rejuvenating factor operably linked to at least one promoter sufficient to drive expression of the at least one rejuvenating factor. A rejuvenating factor can comprise telomerase RNA component (TERC), telomerase associated reverse-transcriptase (TERT), protection of telomeres 1 (POT1), insulin-like growth factor 1 (IGF1), WD repeat containing antisense to TP53 (WRAP53), nuclear protein family A, member 3 (NOP3), heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1), shelterin complex subunit and telomerase recruitment factor (ACD/TPP1), TRF-1 interacting ankyrin-related ADP-ribose polymerase (TNKS), telomeric repeat binding factor 1 (TRF-1), telomeric repeat binding factor 2 (TRF-2), TERF1 interacting nuclear factor 2 (TIN2), telomeric repeat binding factor 2 (Rap1), Dyskerin Pseudouridine Synthase 1 (DKC1), ribonucleoprotein NHP2 or any combination thereof.

The compositions of the present disclosure can be diluted in at least one cell culture medium. In some aspects, the at least one cell culture medium can comprise adjusted Opti-MEM (Opti-MEM with the pH adjusted to 8.2 or Opti-MEM with the pH adjusted to any value in the range between 7.4 and 8.6), non-adjusted Opti-MEM, human serum, fetal bovine serum (FBS), 1× phosphate-buffered saline (PBS) with the pH in the range between of 7.0 and 8.6 or any combination thereof.

In some aspects, any composition of the present disclosure can be packaged into any cellular delivery system known in the art. Cellular delivery systems can include, but are not limited to, adeno-associated virus (AAV; all serotypes, pseudotypes and hybrids), adenovirus, lentivirus, foamy-virus, herpes simplex virus (HSV) particle, retrovirus particle, alphavirus particle, flavivirus particle, rhabdovirus particle, measle virus particle, Newcastle disease virus particle, poxvirus particle, picornavirus particle, nanoparticles, exosomes and any combination thereof.

In some aspects, adeno-associate virus can include, but are not limited to AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV2/1, AAV2/2, AAV2/3, AAV2/4, AAV2/5, AAV2/6, AAV2/7, AAV2/8, AAV2/9, AAV-DJ, AAV-DJ8 or any combination thereof.

The present disclosure provides at least one viral particle, wherein the at least one viral particle comprises any composition of the present disclosure. In some aspects, an at least one viral particle can be an adeno-associated virus (AAV) particle. In some aspects, the at least one viral particle can be an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV2/1, AAV2/2, AAV2/3, AAV2/4, AAV2/5, AAV2/6, AAV2/7, AAV2/8, AAV2/9, AAV-DJ, or AAV-DJ8 particle. In some aspects, the at least one viral particle can be an adenovirus particle. In some aspects, the at least one viral particle can be a foamy-virus particle. In some aspects, the at least one viral particle can be a lentivirus particle. A retrovirus particle can be MMSV or MSCV particle. A lentivirus particle can be HIV-1 or HIV-2 particle. An alphavirus particle can be SFV, SIN, VEE, or M1 particle. A flavivirus particle can be Kunjin virus, West Nile virus, or Dengue virus particle.

The present disclosure provides at least one exosome, microvesicle or liposome, wherein the at least one exosome, microvesicle or liposome comprises any composition of the present disclosure.

The present disclosure provides at least one nanoparticle, wherein the at least one nanoparticle comprises any composition of the present disclosure. In some aspects, a nanoparticle can comprise a liposome, a micelle, a polymer-based nanoparticle, a lipid-polymer based nanoparticle, a metal based nanoparticle, a nanocrystal, a carbon nanotube based nanoparticle or a polymeric micelle. In some aspects, a polymer-based nanoparticle can comprise a multiblock copolymer, a diblock copolymer, a polymeric micelle or a hyperbranched macromolecule. In some aspects, a polymer-based nanoparticle can comprise a multiblock copolymer a diblock copolymer. In some aspects, a polymer-based nanoparticle comprises a poly(lactic-co-glycolic acid) PLGA polymer.

In some aspects, the present disclosure provides a composition comprising: a) at least one modified mRNA molecule comprising a nucleic acid sequence encoding at least a portion of human telomerase reverse transcriptase (hTERT); b) at least one modified mRNA molecule comprising a nucleic acid sequence encoding at least a portion of at least one DNA targeting polypeptide, wherein the at least one DNA targeting polypeptide comprises dCas9 and a VP64-P65-Rta (VPR) molecule; and c) a plurality of guide RNA (gRNA) molecules, wherein at least one gRNA in the plurality is complementary to a nucleic acid sequence located upstream of the endogenous hTERC gene.

Kits

In some aspects, the present disclosure provides a kit comprising any composition of the present disclosure. In some aspects, the present disclosure provides a kit comprising any portion of any composition of the present disclosure. In some aspects, any kit of the present disclosure can be used in any method of the present disclosure.

In a non-limiting example, the present disclosure provides a kit comprising a) at least one first polynucleotide molecule comprising a nucleic acid sequence encoding at least a portion of telomerase reverse transcriptase (TERT); and b) at least one second polynucleotide molecule comprising a nucleic acid sequence encoding at least a portion of at least one DNA targeting polypeptide, wherein the DNA targeting polypeptide increases transcription of telomerase RNA component (TERC).

Rejuvenation Methods of the Present Disclosure

The present disclosure provides a method of rejuvenating at least one cell, the method comprising contacting the at least one cell with at least one composition of the present disclosure. The method can further comprise expanding the at least one cell contacted with the at least one composition of the present disclosure to produce a plurality of rejuvenated cells.

Thus, the present disclosure provides a method of rejuvenating at least one cell, the method comprising contacting the at least one cell with a composition comprising a) at least one first polynucleotide molecule comprising a nucleic acid sequence encoding at least a portion of telomerase reverse transcriptase (TERT); and b) at least one second polynucleotide molecule comprising a nucleic acid sequence encoding at least a portion of at least one DNA targeting polypeptide, wherein the DNA targeting polypeptide increases transcription of telomerase RNA component (TERC).

The present disclosure provides a method of treating and/or preventing a disease in a subject comprising: a) contacting at least one cell with at least one composition of the present disclosure; b) expanding the at least one cell contacted with the at least one composition of the present disclosure to produce a plurality of rejuvenated cells; and c) administering the plurality of rejuvenated cells to the subject.

The present disclosure provides a method of treating and/or preventing a disease in a subject comprising: a) contacting at least one cell with at least one composition of the present disclosure; b) expanding the at least one cell contacted with the at least one composition of the present disclosure to produce a plurality of rejuvenated cells; c) culturing the plurality of rejuvenated cells under conditions sufficient to transform the plurality of rejuvenated cells into at least one tissue or organ; and d) administering the at least one tissue or organ to the subject.

The present disclosure provides a method of producing an in vitro tissue or organ comprising: a) contacting at least one cell with a composition of the present disclosure; b) expanding the at least one cell contacted with the at least one composition of the present disclosure to produce a plurality of rejuvenated cells; c) culturing the plurality of rejuvenated cells under conditions sufficient to transform the plurality of rejuvenated cells into at least one tissue or organ. The at least one tissue or organ can be used for further in vitro testing, including, but not limited to the testing or drugs and/or therapeutic compounds.

The present disclosure provides a method of producing a plurality of rejuvenated cells comprising: a) contacting at least one cell with at least one composition of the present disclosure; b) expanding the at least one cell contacted with the at least one composition of the present disclosure to produce a plurality of rejuvenated cells.

The present disclosure provides a method of producing a plurality of rejuvenated edited cells comprising: a) contacting a plurality of cells with a gene editing system such that at least one gene in the genome of at least one cell in the plurality is edited, thereby producing at least one edited cell; b) isolating the at least one edited cell; c) contacting the isolated at least one edited cell with at least one composition of the present disclosure; and d) expanding the at least one cell contacted with the at least one composition of the present disclosure to produce a plurality of rejuvenated edited cell.

The present disclosure provides a method of treating and/or preventing a disease in a subject comprising: a) contacting a plurality of cells with a gene editing system such that at least one gene in the genome of at least one cell in the plurality is edited, thereby producing at least one edited cell; b) isolating the at least one edited cell; c) contacting the isolated at least one edited cell with at least one composition of the present disclosure; d) expanding the at least one cell contacted with the at least one composition of the present disclosure to produce a plurality of rejuvenated edited cells; and e) administering to the subject the plurality of rejuvenated edited cells.

In some aspects, the present disclosure provides a method of treating epidermolysis bullosa (EB) in a subject comprising: a) contacting a plurality of cells comprising keratinocytes, dermal fibroblasts, mesenchymal stem/stromal cells or any combination thereof with a gene editing system such that at least one gene in the genome of at least one cell in the plurality is edited, thereby producing at least one edited cell; b) isolating the at least one edited cell; c) contacting the isolated at least one edited cell with at least one composition of the present disclosure; d) expanding the at least one cell contacted with the at least one composition of the present disclosure to produce a plurality of rejuvenated edited cells; and e) administering to the subject the plurality of rejuvenated edited cells.

In some aspects, the present disclosure provides a method of rejuvenating at least one cell in a subject comprising administering to the subject at least one therapeutically effective amount of at least one composition of the present disclosure. In some aspects, the present disclosure provides a method of rejuvenating at least one cell in a subject comprising administering to the subject at least one therapeutically effective amount of at least one portion of at least one composition of the present disclosure.

In some aspects, the present disclosure provides a method of rejuvenating at least one subject comprising administering at least one therapeutically effective amount of at least one composition of the present disclosure. In some aspects, the present disclosure provides a method of rejuvenating at least one subject comprising administering at least one therapeutically effective amount of at least one portion of at least one composition of the present disclosure.

In some aspects of the methods of the present disclosure, contacting at least one cell with at least one composition of the present disclosure can comprise contacting the at least one cell with a first portion of the at least one composition of the present disclosure and then contacting the at least one cell with a second portion of the least one composition of the present disclosure at least about 1 hour, or at least about 2 hours, or at least about 3 hours, or at least about 4 hours, or at least about 5 hours, or at least about 6 hours, or at least about 7 hours, or at least about 8 hours, or at least about 9 hours, or at least about 10 hours, or at least about 11 hours, or at least about 12 hours, or at least about 16 hours, or at least about 20 hours, or at least about 24 hours, or at least about 28 hours, or at least about 32 hours, or at least about 36 hours, or at least about 40 hours, or at least about 44 hours, or at least about 48 hours, or at least about 52 hours, or at least about 56 hours, or at least about 60 hours, or at least about 64 hours, or at least about 68 hours, or at least about 72 hours, or at least about 76 hours, or at least about 80 hours, or at least about 84 hours, or at least about 88 hours, or at least about 92 hours, or at least about 96 hours after contacting the at least one cell with the first portion of the at least one composition of the present disclosure.

Thus, contacting at least one cell with at least one composition of the present disclosure can comprise: a) contacting the at least one cell with at least one first polynucleotide molecule comprising a nucleic acid sequence encoding at least a portion of telomerase reverse transcriptase (TERT); and b) contacting the at least one cell with at least one second polynucleotide molecule comprising a nucleic acid sequence encoding at least a portion of at least one DNA targeting polypeptide, wherein the DNA targeting polypeptide increases transcription of telomerase RNA component (TERC), at least about 24 hours after step (a). Optionally, steps (a) and step (b) can be repeated about every 1, or about every 2, or about every 3, or about every 4, or about every 5, or about every 6, or about every 7, or about every 8, or about every 9, or about every 10 days.

In some aspects of the methods of the present disclosure, contacting at least one cell with at least one composition of the present disclosure can further comprise pretreating the at least one cell. In some aspects, pretreating a cell can comprise contacting the at least one cell with at least one first polynucleotide molecule comprising a nucleic acid sequence encoding at least a portion of telomerase reverse transcriptase (TERT) once about every 4 hours, or about every 8 hours, or about every 12 hours, or about every 16 hours, or about every 20 hours, or about every 24 hours, or about every 28 hours, or about every 32 hours, or about every 36 hours, or about every 40 hours, or about every 44 hours, or about every 48 hours. In some aspects, the at least one cell can be pretreated for at least about 2, or at least about 4, or at least about 6, or at least about 8, or at least about 10 days.

In some aspects, contacting at least one cell with at least one composition of the present disclosure can comprise: a) contacting the at least one cell with at least one first polynucleotide molecule comprising a nucleic acid sequence encoding at least a portion of telomerase reverse transcriptase (TERT) and at least one second polynucleotide molecule comprising a nucleic acid sequence encoding at least a portion of at least one DNA targeting polypeptide, wherein the DNA targeting polypeptide increases transcription of telomerase RNA component (TERC); and b) repeating step (a) about every 1, or about every 2, or about every 3, or about every 4, or about every 5, or about every 6, or about every 7, or about every 8, or about every 9, or about every 10 days.

Exemplary transfection regimes are shown in FIGS. 8, 10 and 11.

In some aspects of the methods of the present disclosure, contacting at least one cell with a composition of the present disclosure can comprise transfection. In some aspects, transfection can comprise the use of lipofectamine. In some aspects, transfection can comprise any standard transfection method known in the art. In some aspects of the methods of the present disclosure, contacting at least one cell with a composition of the present disclosure can comprise electroporation.

In some aspects of the methods of the present disclosure, contacting at least one cell can comprise transfection, transduction, electroporation, nucleofection, at least one cell-penetrating peptide or any combination thereof.

In some aspects of the methods of the present disclosure, contacting at least one cell with a composition of the present disclosure can comprise nucleofection. In some aspects, nucleofection can comprise any standard nucleofection method known in the art.

In some aspects of the methods of the present disclosure, contacting at least one cell with a composition of the present disclosure can comprise contacting the cell with at least one cell-penetrating peptides. In some aspects, a cell-penetrating peptide can be an HIV-derived TAT protein. In some aspects, a cell-penetrating peptide can comprise polyarginine. Without wishing to be bound by theory, the at least one cell-penetrating peptide can aid in the delivery of a protein or a RNP complex of the present disclosure to the cytoplasm of a target cell.

In some aspects, at least one composition or at least one portion of at least one composition of the present disclosure can be administered to a subject orally, nasally, transdermally, pulmonary, inhalationally, buccally, sublingually, intraperitoneally, subcutaneously, intramuscularly, intravenously, rectally, intrapleurally, intrathecally and/or parenterally.

In some aspects of the methods of the present disclosure, expanding at least one cell can comprise culturing the at least one cell using adjusted Opti-MEM, non-adjusted Opti-MEM, human serum, fetal bovine serum (FBS) or any combination thereof.

In some aspect of the methods of the present disclosure, rejuvenating at least one cell can comprise increasing the expression of TERC in the at least one cell. In some aspects, rejuvenating at least one cell can comprise increasing the expression of TERC by at least about 1%, or at least about 5%, or at least about 10%, or at least about 15%, or at least about 20%, or at least about 25%, or at least about 30%, or at least about 35%, or at least about 40%, or at least about 45%, or at least about 50%, or at least about 55%, or at least about 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 100%, or at least about 150%, or at least about 200%, or at least about 250%, or at least about 300%, or at least about 350%, or at least about 400%, or at least about 450%, or at least about 500%, or at least about 550%, or at least about 600%, or at least about 650%, or at least about 700%, or at least about 750%, or at least about 800%, or at least about 850%, or at least about 900%, or at least about 950%, or at least about 1000%, or at least about 10,000%, or at least about 100,000%, or at least about 1,000,000%, or at least about 10,000,000%, or at least about 100,000,000%. In some aspects, rejuvenating at least one cell can comprise increasing the expression of TERC in the at least one cell such that the expression level of TERC after contacting the at least one cell with at least one composition of the present disclosure is at least about 0.5 times, or at least about 1.0 times, or at least about 1.5 times, or at least about 2.0 times, or at least about 2.5 times, or at least about 3.0 times, or at least about 3.5 times, or at least about 4.0 times, or at least about 4.5 times, or at least about 5.0 times, or at least about 5.5 times, or at least about 6.0 times, or at least about 6.5 times, or at least about 7.0 times, or at least about 7.5 times, or at least about 8.0 times, or at least about 8.5 times, or at least about 9.0 times, or at least about 9.5 times, or at least about 10.0 times, or at least about 25 times, or at least about 50 times, or at least about 75 times, or at least about 100 times, or at least about 1000 times, or at least about 10,000 times, or at least about 20,000 times, or at least about 30,000 times, or at least about 40,000 times, or at least about 50,000 times, or at least about 60,000 times, or at least about 70,000 times, or at least about 80,000 times, or at least about 90,000, or at least about 100,000 times greater as compared to the expression level of TERC prior to contacting the at least one cell with the at least one composition of the present disclosure.

In some aspects of the methods of the present disclosure, rejuvenating at least one cell can comprise increasing the expression of TERC in the at least one cell such that the expression level of TERC is at least about the same as, or at least about 1.5 times, or at least about 2.0 times, or at least about 2.5 times, or at least about 3.0 times, or at least about 3.5 times, or at least about 4.0 times, or at least about 4.5 times, or at least about 5.0 times, or at least about 5.5 times, or at least about 6.0 times, or at least about 6.5 times, or at least about 7.0 times, or at least about 7.5 times, or at least about 8.0 times, or at least about 8.5 times, or at least about 9.0 times, or at least about 9.5 times, or at least about 10.0 times, or at least about 25 times, or at least about 50 times, or at least about 75 times, or at least about 100 times, or at least about 1000 times, or at least about 10,000 times, or at least about 20,000 times, or at least about 30,000 times, or at least about 40,000 times, or at least about 50,000 times, or at least about 60,000 times, or at least about 70,000 times, or at least about 80,000 times, or at least about 90,000, or at least about 100,000 times the expression of TERC in a control cell.

In some aspect of the methods of the present disclosure, rejuvenating at least one cell can comprise increasing the expression of TERT in the at least one cell. In some aspects, rejuvenating at least one cell can comprise increasing the expression of TERT by at least about 1%, or at least about 5%, or at least about 10%, or at least about 15%, or at least about 20%, or at least about 25%, or at least about 30%, or at least about 35%, or at least about 40%, or at least about 45%, or at least about 50%, or at least about 55%, or at least about 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 100%, or at least about 150%, or at least about 200%, or at least about 250%, or at least about 300%, or at least about 350%, or at least about 400%, or at least about 450%, or at least about 500%, or at least about 550%, or at least about 600%, or at least about 650%, or at least about 700%, or at least about 750%, or at least about 800%, or at least about 850%, or at least about 900%, or at least about 950%, or at least about 1000% or at least about 10,000%, or at least about 100,000%, or at least about 1,000,000%, or at least about 10,000,000%, or at least about 100,000,000%. In some aspects, rejuvenating at least one cell can comprise increasing the expression of TERT in the at least one cell such that the expression level of TERT after contacting the at least one cell with at least one composition of the present disclosure is at least about 0.5 times, or at least about 1.0 times, or at least about 1.5 times, or at least about 2.0 times, or at least about 2.5 times, or at least about 3.0 times, or at least about 3.5 times, or at least about 4.0 times, or at least about 4.5 times, or at least about 5.0 times, or at least about 5.5 times, or at least about 6.0 times, or at least about 6.5 times, or at least about 7.0 times, or at least about 7.5 times, or at least about 8.0 times, or at least about 8.5 times, or at least about 9.0 times, or at least about 9.5 times, or at least about 10.0 times, or at least about 25 times, or at least about 50 times, or at least about 75 times, or at least about 100 times, or at least about 1000 times, or at least about 10,000 times, or at least about 20,000 times, or at least about 30,000 times, or at least about 40,000 times, or at least about 50,000 times, or at least about 60,000 times, or at least about 70,000 times, or at least about 80,000 times, or at least about 90,000, or at least about 100,000 times greater as compared to the expression level of TERT prior to contacting the at least one cell with the at least one composition of the present disclosure.

In some aspects of the methods of the present disclosure, rejuvenating at least one cell can comprise increasing the expression of TERT in the at least one cell such that the expression level of TERT is at least about the same as, or at least about 1.5 times, or at least about 2.0 times, or at least about 2.5 times, or at least about 3.0 times, or at least about 3.5 times, or at least about 4.0 times, or at least about 4.5 times, or at least about 5.0 times, or at least about 5.5 times, or at least about 6.0 times, or at least about 6.5 times, or at least about 7.0 times, or at least about 7.5 times, or at least about 8.0 times, or at least about 8.5 times, or at least about 9.0 times, or at least about 9.5 times, or at least about 10.0 times, or at least about 25 times, or at least about 50 times, or at least about 75 times, or at least about 100 times, or at least about 1000 times, or at least about 10,000 times, or at least about 20,000 times, or at least about 30,000 times, or at least about 40,000 times, or at least about 50,000 times, or at least about 60,000 times, or at least about 70,000 times, or at least about 80,000 times, or at least about 90,000, or at least about 100,000 times the expression of TERT in a control cell.

In some aspects of the methods of the present disclosure, rejuvenating at least one cell can comprise increasing the total number of population doublings exhibited by the at least one cell. In some aspects, rejuvenating at least one cell can comprise increasing the total number of population doublings exhibited by the at least one cell by at least about 1%, or at least about 5%, or at least about 10%, or at least about 15%, or at least about 20%, or at least about 25%, or at least about 30%, or at least about 35%, or at least about 40%, or at least about 45%, or at least about 50%, or at least about 55%, or at least about 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 100%, or at least about 150%, or at least about 200%, or at least about 250%, or at least about 300%, or at least about 350%, or at least about 400%, or at least about 450%, or at least about 500%, or at least about 550%, or at least about 600%, or at least about 650%, or at least about 700%, or at least about 750%, or at least about 800%, or at least about 850%, or at least about 900%, or at least about 950%, or at least about 1,000%, or at least about 2,000%, or at least about 3,000%, or at least about 4,000%, or at least about 5,000%, or at least about 6,000%, or at least about 7,000%, or at least about 8,000%, or at least about 9,000%, or at least about 10,000%, or at least about 20,000%, or at least about 30,000%, or at least about 40,000%, or at least about 50,000%, or at least about 60,000%, or at least about 70,000%, or at least about 80,000%, or at least about 90,000%, or at least about 100,000%.

In some aspects, rejuvenating at least one cell can comprise increasing the total number of population doublings such that the number of population doublings exhibited by the at least one cell is at least about 1.5 times, or at least about 2.0 times, or at least about 2.5 times, or at least about 3.0 times, or at least about 3.5 times, or at least about 4.0 times, or at least about 4.5 times, or at least about 5.0 times, or at least about 5.5 times, or at least about 6.0 times, or at least about 6.5 times, or at least about 7.0 times, or at least about 7.5 times, or at least about 8.0 times, or at least about 8.5 times, or at least about 9.0 times, or at least about 9.5 times, or at least about 10.0 times, or at least about 25 times, or at least about 50 times, or at least about 75 times, or at least about 100 times, or at least about 200 times, or at least about 300 times, or at least about 400 times, or at least about 500 times, or at least about 600 times, or at least about 700 times or at least about 800 times, or at least about 900 times, or at least about 1,000 times the total number of population doublings exhibited by at least one control cell.

In some aspects of the methods of the present disclosure, rejuvenating at least one cell can comprise increasing the length of telomeres in the at least one cell. In some aspects, rejuvenating at least one cell can comprising increasing the length of telomeres in the at least one cell by at least about 1%, or at least about 5%, or at least about 10%, or at least about 15%, or at least about 20%, or at least about 25%, or at least about 30%, or at least about 35%, or at least about 40%, or at least about 45%, or at least about 50%, or at least about 55%, or at least about 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 100%, or at least about 150%, or at least about 200%, or at least about 250%, or at least about 300%, or at least about 350%, or at least about 400%, or at least about 450%, or at least about 500%, or at least about 550%, or at least about 600%, or at least about 650%, or at least about 700%, or at least about 750%, or at least about 800%, or at least about 850%, or at least about 900%, or at least about 950%, or at least about 1000%, or at leaset about 2000%, or at least about 3000%, or at least about 4000%, or at least about 5000%, or at least about 6000%, or at least about 7000%, or at least about 8,000%, or at least about 9,000%, or at least about 10,000%, or at least about 20,000%, or at least about 30,000%, or at least about 40,000%, or at least about 50,000%, or at least about 60,000%, or at least about 70,000%, or at least about 80,000%, or at least about 90,000%, or at least about 100,000%.

In some aspects, rejuvenating at least one cell can comprise increasing the length of telomeres in the at least one cell such that the length of the telomeres in the at least one cell is the same as, or at least about 1.5 times, or at least about 2.0 times, or at least about 2.5 times, or at least about 3.0 times, or at least about 3.5 times, or at least about 4.0 times, or at least about 4.5 times, or at least about 5.0 times, or at least about 5.5 times, or at least about 6.0 times, or at least about 6.5 times, or at least about 7.0 times, or at least about 7.5 times, or at least about 8.0 times, or at least about 8.5 times, or at least about 9.0 times, or at least about 9.5 times, or at least about 10.0 times, or at least about 25 times, or at least about 50 times, or at least about 75 times, or at least about 100 times, or at least about 200 times, or at least about 300 times, or at least about 400 times, or at least about 500 times, or at least about 600 times, or at least about 700 times, or at least about 800 times, or at least about 900 times or at least about 1,000 times the length of telomeres in at least one control cell.

In some aspects of the methods of the present disclosure, rejuvenating at least one cell can comprise increasing the mitochondrial DNA copy number in the at least one cell. In some aspects, rejuvenating at least one cell can comprise increasing the mitochondrial DNA copy number in the at least one cell by a at least about 1%, or at least about 5%, or at least about 10%, or at least about 15%, or at least about 20%, or at least about 25%, or at least about 30%, or at least about 35%, or at least about 40%, or at least about 45%, or at least about 50%, or at least about 55%, or at least about 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 100%, or at least about 150%, or at least about 200%, or at least about 250%, or at least about 300%, or at least about 350%, or at least about 400%, or at least about 450%, or at least about 500%, or at least about 550%, or at least about 600%, or at least about 650%, or at least about 700%, or at least about 750%, or at least about 800%, or at least about 850%, or at least about 900%, or at least about 950%, or at least about 1,000%, or at least about 2,000%, or at least about 3,000%, or at least about 4,000%, or at least about 5,000%, or at least about 6,000%, or at least about 7,000%, or at least about 8,000%, or at least about 9,000%, or at least about 10,000%, or at least about 20,000%, or at least about 30,000%, or at least about 40,000%, or at least about 50,000%, or at least about 60,000%, or at least about 70,000%, or at least about 80,000%, or at least about 90,000%, or at least abouat 100,000%.

In some aspects, rejuvenating at least one cell can comprise increasing the mitochondrial DNA copy number in the at least one cell such that the mitochondrial DNA copy number is the same as, or at least about 1.5 times, or at least about 2.0 times, or at least about 2.5 times, or at least about 3.0 times, or at least about 3.5 times, or at least about 4.0 times, or at least about 4.5 times, or at least about 5.0 times, or at least about 5.5 times, or at least about 6.0 times, or at least about 6.5 times, or at least about 7.0 times, or at least about 7.5 times, or at least about 8.0 times, or at least about 8.5 times, or at least about 9.0 times, or at least about 9.5 times, or at least about 10.0 times, or at least about 25 times, or at least about 50 times, or at least about 75 times, or at least about 100 times, or at least about 200 times, or at least about 300 times, or at least about 400 times, or at least about 500 times, or at least about 600 times, or at least about 700 times, or at least about 800 times, or at least about 900 times, or at least about 1000 times the mitochondrial DNA copy number in at least one control cell.

In some aspects of the methods of the present disclosure, rejuvenating at least one cell can comprise increasing the amount of mitochondrial DNA in the at least one cell. In some aspects, rejuvenating at least one cell can comprise increasing the amount of mitochondrial DNA in the at least one cell by at least about 1%, or at least about 5%, or at least about 10%, or at least about 15%, or at least about 20%, or at least about 25%, or at least about 30%, or at least about 35%, or at least about 40%, or at least about 45%, or at least about 50%, or at least about 55%, or at least about 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 100%, or at least about 150%, or at least about 200%, or at least about 250%, or at least about 300%, or at least about 350%, or at least about 400%, or at least about 450%, or at least about 500%, or at least about 550%, or at least about 600%, or at least about 650%, or at least about 700%, or at least about 750%, or at least about 800%, or at least about 850%, or at least about 900%, or at least about 950%, or at least about 1,000%, or at least about 2,000%, or at least about 3,000%, or at least about 4,000%, or at least about 5,000%, or at least about 6,000%, or at least about 7,000%, or at least about 8,000%, or at least about 9,000%, or at least about 10,000%, or at least about 20,000%, or at least about 30,000%, or at least about 40,000%, or at least about 50,000%, or at least about 60,000%, or at least about 70,000%, or at least about 80,000%, or at least about 90,000%, or at least abouat 100,000%.

In some aspects, rejuvenating at least one cell can comprise increasing the amount of mitochondrial DNA in the at least one cell such that the amount of mitochondrial DNA in the at least one cell is the same as, or at least about 1.5 times, or at least about 2.0 times, or at least about 2.5 times, or at least about 3.0 times, or at least about 3.5 times, or at least about 4.0 times, or at least about 4.5 times, or at least about 5.0 times, or at least about 5.5 times, or at least about 6.0 times, or at least about 6.5 times, or at least about 7.0 times, or at least about 7.5 times, or at least about 8.0 times, or at least about 8.5 times, or at least about 9.0 times, or at least about 9.5 times, or at least about 10.0 times, or at least about 25 times, or at least about 50 times, or at least about 75 times, or at least about 100 times, or at least about 200 times, or at least about 300 times, or at least about 400 times, or at least about 500 times, or at least about 600 times, or at least about 700 times, or at least about 800 times, or at least about 900 times, or at least about 1000 times the amount of mitochondrial DNA in at least one control cell.

In some aspects of the methods of the present disclosure, rejuvenating at least one cell can comprise increasing the number of mitochondria in the at least one cell. In some aspects, rejuvenating at least one cell can comprise increasing the number of mitochondria in the at least one cell by at least about 1%, or at least about 5%, or at least about 10%, or at least about 15%, or at least about 20%, or at least about 25%, or at least about 30%, or at least about 35%, or at least about 40%, or at least about 45%, or at least about 50%, or at least about 55%, or at least about 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 100%, or at least about 150%, or at least about 200%, or at least about 250%, or at least about 300%, or at least about 350%, or at least about 400%, or at least about 450%, or at least about 500%, or at least about 550%, or at least about 600%, or at least about 650%, or at least about 700%, or at least about 750%, or at least about 800%, or at least about 850%, or at least about 900%, or at least about 950%, or at least about 1,000%, or at least about 2,000%, or at least about 3,000%, or at least about 4,000%, or at least about 5,000%, or at least about 6,000%, or at least about 7,000%, or at least about 8,000%, or at least about 9,000%, or at least about 10,000%, or at least about 20,000%, or at least about 30,000%, or at least about 40,000%, or at least about 50,000%, or at least about 60,000%, or at least about 70,000%, or at least about 80,000%, or at least about 90,000%, or at least abouat 100,000%.

In some aspects, rejuvenating at least one cell can comprise increasing the number of mitochondria in the at least one cell such that the number of mitochondria is the same as, or at least about 1.5 times, or at least about 2.0 times, or at least about 2.5 times, or at least about 3.5 times, or at least about 4.0 times, or at least about 4.5 times, or at least about 5.0 times, or at least about 5.5 times, or at least about 6.0 times, or at least about 6.5 times, or at least about 7.0 times, or at least about 7.5 times, or at least about 8.0 times, or at least about 8.5 times, or at least about 9.0 times, or at least about 9.5 times, or at least about 10.0 times, or at least about 25 times, or at least about 50 times, or at least about 75 times, or at least about 100 times, or at least about 200 times, or at least about 300 times, or at least about 400 times, or at least about 500 times, or at least about 600 times, or at least about 700 times, or at least about 800 times, or at least about 900 times, or at least about 1,000 times the amount of mitochondria in at least one control cell.

In some aspects of the methods of the present disclosure, rejuvenating at least one cell can comprise increasing the migration activity of the at least one cell. In some aspects, rejuvenating at least one cell can comprise increasing the migration activity of the at least one cell by at least about 1%, or at least about 5%, or at least about 10%, or at least about 15%, or at least about 20%, or at least about 25%, or at least about 30%, or at least about 35%, or at least about 40%, or at least about 45%, or at least about 50%, or at least about 55%, or at least about 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 100%, or at least about 150%, or at least about 200%, or at least about 250%, or at least about 300%, or at least about 350%, or at least about 400%, or at least about 450%, or at least about 500%, or at least about 550%, or at least about 600%, or at least about 650%, or at least about 700%, or at least about 750%, or at least about 800%, or at least about 850%, or at least about 900%, or at least about 950%, or at least about 1,000%, or at least about 2,000%, or at least about 3,000%, or at least about 4,000%, or at least about 5,000%, or at least about 6,000%, or at least about 7,000%, or at least about 8,000%, or at least about 9,000%, or at least about 10,000%, or at least about 20,000%, or at least about 30,000%, or at least about 40,000%, or at least about 50,000%, or at least about 60,000%, or at least about 70,000%, or at least about 80,000%, or at least about 90,000%, or at least abouat 100,000%.

In some aspects, rejuvenating at least one cell can comprise increasing the migration activity of the at least one cell such that the migration activity of the at least one cell is the same as, or at least 1.5 times, or at least about 2.0 times, or at least about 2.5 times, or at least about 3.5 times, or at least about 4.0 times, or at least about 4.5 times, or at least about 5.0 times, or at least about 5.5 times, or at least about 6.0 times, or at least about 6.5 times, or at least about 7.0 times, or at least about 7.5 times, or at least about 8.0 times, or at least about 8.5 times, or at least about 9.0 times, or at least about 9.5 times, or at least about 10.0 times, or at least about 25 times, or at least about 50 times, or at least about 75 times, or at least about 100 times, or at least about 200 times, or at least about 300 times, or at least about 400 times, or at least about 500 times, or at least about 600 times, or at least about 700 times, or at least about 800 times, or at least about 900 times, or at least about 1000 times the migration activity of at least one control cell.

In some aspects, rejuvenating at least one cell can comprise restoring the young-like state of thiol group oxidation on at least one protein in the at least one cell. In a non-limiting example, rejuvenating can comprise increasing the thiol group oxidation of at least one protein in the at least one cell such that the thiol group oxidation of the at least one protein in the at least one cell is comparable to the thiol group oxidation of the same protein in a young cell. In a non-limiting example, rejuvenating can comprise decreasing the thiol group oxidation of at least one protein in the at least one cell such that the thiol group oxidation of the at least one protein in the at least one cell is comparable to the thiol group oxidation of the same protein in a young cell.

In some aspects, rejuvenating at least one cell can comprise decreasing the thiol group oxidation of at least one protein in the at least one cell by at least about 1%, or at least about 5%, or at least about 10%, or at least about 15%, or at least about 20%, or at least about 25%, or at least about 30%, or at least about 35%, or at least about 40%, or at least about 45%, or at least about 50%, or at least about 55%, or at least about 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 100%. In some aspects, the at least one protein can be EIF2S1, TM9F3 or USP14.

In some aspects, rejuvenating at least one cell can comprise increasing the thiol group oxidation of at least one protein in the at least one cell by at least about 1%, or at least about 5%, or at least about 10%, or at least about 15%, or at least about 20%, or at least about 25%, or at least about 30%, or at least about 35%, or at least about 40%, or at least about 45%, or at least about 50%, or at least about 55%, or at least about 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 100%, or at least about 150%, or at least about 200%, or at least about 250%, or at least about 300%, or at least about 350%, or at least about 400%, or at least about 450%, or at least about 500%, or at least about 550%, or at least about 600%, or at least about 650%, or at least about 700%, or at least about 750%, or at least about 800%, or at least about 850%, or at least about 900%, or at least about 950%, or at least about 1,000%, or at least about 2,000%, or at least about 3,000%, or at least about 4,000%, or at least about 5,000%, or at least about 6,000%, or at least about 7,000%, or at least about 8,000%, or at least about 9,000%, or at least about 10,000%, or at least about 20,000%, or at least about 30,000%, or at least about 40,000%, or at least about 50,000%, or at least about 60,000%, or at least about 70,000%, or at least about 80,000%, or at least about 90,000%, or at least abouat 100,000%. In some aspects, the at least one protein can be IGFB5.

In some aspects of the methods of the present disclosure, rejuvenating at least one cell can comprise reducing senescence-associated DNA methylation in the at least one cell. In some aspects, reducing senescence-associated DNA methylation in the at least one cell can comprise reducing DNA methylation at least one genomic location which is associated with senescence-related methylation. In some aspects, the at least one genomic location can be cg09780241, cg05099537, cg24541426, cg04316624, cg13180312, cg13316854, cg15726154, cg21507095, cg01697719 or any combination thereof.

In some aspects, rejuvenating at least one cell can comprise reducing DNA methylation at least one genomic location by at least about 1%, or at least about 5%, or at least about 10%, or at least about 15%, or at least about 20%, or at least about 25%, or at least about 30%, or at least about 35%, or at least about 40%, or at least about 45%, or at least about 50%, or at least about 55%, or at least about 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 100%.

In some aspects, an at least one control cell can comprise a cell that has not been contacted with a composition of the present disclosure. In some aspects, an at least one control cell can comprise a cell that has not been contacted with a composition of the present disclosure but has otherwise been grown under the same conditions as at least one cell contacted with a composition of the present disclosure. In some aspects, an at least one control cell can be a dermal fibroblast isolated from a human subject that is 50 years old. In some aspects, an at least one control cell can be a neonatal human epidermal keratinocyte (HEKn). In some aspects, an at least one control cell can be an induced pluripotent stem cell (iPSC).

In some aspects, editing at least one cell can comprise the correction of at least one gene in the at least one cell, the knockout of at least one gene in the at least one cell, the insertion of at least one DNA sequence into the genome of the at least one cell, the deletion of at least one DNA sequence in the genome of the at least one cell or any combination thereof. In some aspects, a gene editing system can comprise any system known in the art for modifying the genome of a target cell, including, but not limited to CRISPR methods, viral methods, etc.

An at least one cell can be obtained and/or isolated from a subject. In some aspects, an at least one cell can be any somatic cell. In some aspects, an at least one cell can be a fibroblast, a keratinocyte, a mesenchymal stem/stromal cell, a peripheral blood mononuclear cell, a chimeric antigen receptor T cell (CAR-T cell), an endothelial cell, a chondrocyte, a muscle stem cell, a neural stem cell, a hepatocyte, a limbal stem cell, a retinal pigmented epithelial cell, a hematopoietic stem cell, a macrophage, a cardiomyocyte, a pancreatic cell, a β-cell or any combination thereof.

In some aspects, an at least one cell can be an Exocrine secretory epithelial cell, a Brunner's gland cell, an insulated goblet cell of the respiratory and digestive tracts, a stomach foveolar cell, a chief cell, a parietal cell, a pancreatic acinar cell, a paneth cell of small intestine, a Type II pneumocyte of lung, a club cell of lung, a barrier cell, a type I pneumocyte, a gall bladder epithelial cell, a centroacinar cell, an intercalated duct cell, an intestinal brush border cell, a hormone-secreting cell, an enteroendocrine cell, a K cell, an L cell, an I cell, a G cell, an Enterochromaffin cell, an Enterochromaffin-like cell, an N cell, an S cell, a D cell, a Mo cell (or M cell), a Thyroid gland cell, a Thyroid epithelial cell, a Parafollicular cell, a Parathyroid gland cell, a Parathyroid chief cell, an Oxyphil cell, a Pancreatic islets (islets of Langerhans), an Alpha cell, a Beta cell, a Delta cell, an Epsilon cell, a PP cell (gamma cell), an Exocrine secretory epithelial cell, a Salivary gland mucous cell, a Salivary gland serous cell, a Von Ebner's gland cell, a Mammary gland cell, a Lacrimal gland cell, a Ceruminous gland cell, an Eccrine sweat gland dark cell, a Eccrine sweat gland clear cell, an Apocrine sweat gland cell, a Gland of Moll cell, a Sebaceous gland cell, a Bowman's gland cell, an Anterior/Intermediate pituitary cell, a Corticotrope, a Gonadotrope, a Lactotrope, a Melanotrope, a Somatotrope, a thyrotrope, a magnocellular neurosecretory cell, a Parvocellular neurosecretory cell, a Chromaffin cell, an Epithelial cell, a Keratinocyte, an Epidermal basal cell, a Melanocyte, a Trichocyte, a hair shaft cell, a Cortical hair shaft cell, a Cuticular hair shaft cell, a Huxley's layer hair root sheath cell, a Henle's layer hair root sheath cell, an Outer root sheath hair cell, a Surface epithelial cell, a basal cell (stem cell), an Intercalated duct cell, a Striated duct cell, a Lactiferous duct cell, an Ameloblast, an Oral cell, an Odontoblast, a Cementoblast, a neuron, an Auditory inner hair cell, an auditory outer hair cell, a Basal cell of olfactory epithelium cell, a Cold-sensitive primary sensory neuron, a Heat-sensitive primary sensory neuron, a Merkel cell, a Olfactory receptor neuron, a Pain-sensitive primary sensory neuron, a Photoreceptor cell, a Photoreceptor rod cell, a Photoreceptor blue-sensitive cone cell, a Photoreceptor green-sensitive cone cell, a Photoreceptor red-sensitive cone cell, a Proprioceptive primary sensory neuron, a Touch-sensitive primary sensory neuron, a Chemoreceptor glomus cell, an Outer hair cell, an Inner hair cell, a Taste receptor cell, an autonomic neuron, a Cholinergic neuron, a Adrenergic neural cell, a Peptidergic neural cell, an Inner pillar cell, an Outer pillar cell, an Inner phalangeal cell, an Outer phalangeal cell, a Border cell, a Hensen's cell, a Vestibular apparatus supporting cell, a Taste bud supporting cell, a Olfactory epithelium supporting cell, a Schwann cell, a Satellite glial cell, a Enteric glial cell, a glial cell, an interneuron, a Basket cell, a Cartwheel cell, a Stellate cell, a Golgi cell, a Granule cell, a Lugaro cell, a Unipolar brush cell, a Martinotti cell, a Chandelier cell, a Cajal-Retzius cell, a Double-bouquet cell, a Neurogliaform cell, a Retina horizontal cell, an Amacrine cell, a Spinal interneuron, a Renshaw cell, a principal cell, a Spindle neuron, a Fork neuron, a Pyramidal cell, a Place cell, a Grid cell, a Speed cell, a Head direction cell, a Betz cell, a Stellate cell, a Boundary cell, a Bushy cell, a Purkinje cell, a Medium spiny neuron, a Astrocyte (various types), a Oligodendrocyte, a Ependymal cell, a Tanycytes, a Pituicyte, a Lens cell, an Anterior lens epithelial cell, a Crystallin-containing lens fiber cell, a Adipocytes: White fat cell, a Brown fat cell, a Liver lipocyte, a Theca interna cell, a Corpus luteum cell, a Granulosa lutein cell, a Theca lutein cell, a Leydig cell of testes secreting testosterone, a Seminal vesicle cell, a Prostate gland cell, a Bulbourethral gland cell, a Bartholin's gland cell, a Gland of Littre cell, a Uterus endometrium cell, a Juxtaglomerular cell, a Macula densa cell of kidney, a Peripolar cell of kidney, a Mesangial cell of kidney, a barrier cell, a Parietal epithelial cell, a Podocyte, a Proximal tubule brush border cell, a Loop of Henle thin segment cell, a Kidney distal tubule cell, a Kidney collecting duct cell Principal cell, a Intercalated cell, a Transitional epithelium, a Duct cell, a Efferent ducts cell, a Epididymal principal cell, a Epididymal basal cell, a Endothelial cell, a Planum semilunatum epithelial cell, a interdental epithelial cell, a Corneal fibroblasts, a Tendon fibroblasts, a Bone marrow reticular tissue fibroblasts, an Other nonepithelial fibroblasts, a Pericyte Hepatic stellate cell (Ito cell), a Nucleus pulposus cell of intervertebral disc, a Hyaline cartilage chondrocyte, a Fibrocartilage chondrocyte, an Elastic cartilage chondrocyte, a Osteoblast/osteocyte, a Osteoprogenitor cell, a Hyalocyte of vitreous body of eye, a Stellate cell of perilymphatic space of ear, a Pancreatic stellate cell, a Skeletal muscle cell, a Red skeletal muscle cell, a White skeletal muscle cell, an Intermediate skeletal muscle cell, a Nuclear bag cell of muscle spindle, a Nuclear chain cell of muscle spindle, a Myosatellite cell, a Cardiac muscle cell, a Cardiac muscle cell, a SA node cell, a Purkinje fiber cell, a Smooth muscle cell, a Myoepithelial cell, a Erythrocyte, a Megakaryocyte, a Platelets, a Monocyte, a Connective tissue macrophage, a Epidermal Langerhans cell, a Osteoclast, a Dendritic cell, a Microglial cell, a Neutrophil granulocyte, a myeloblast, a promyelocyte, a myelocyte, a metamyelocyte, a Eosinophil granulocyte, a Basophil granulocyte, a Mast cell, a Helper T cell, a Suppressor T cell, a Cytotoxic T cell, a Natural killer T cell, a B cell, a Plasma cell, a Natural killer cell, a Hematopoietic stem cell, a Germ cell, a Oogonium/Oocyte, a Spermatid, a Spermatocyte, a Spermatogonium cell, a Spermatozoon, a Nurse cell, a Granulosa cell, a Sertoli cell, a Epithelial reticular cell, a Interstitial cell, a Interstitial kidney cell or any combination thereof.

In some aspects, a disease can comprise inflammatory disorder, an autoimmune disease, a degenerative disease, cardiovascular disease, ischemic disease, cancer, a genetic disease, a metabolic disorder, idiopathic disorder or any combination thereof. In some aspects, a disease can comprise any medical disorder, including, but not limited to those medical disorders initiated by direct tissue injury (e.g., burns, trauma, decubitus ulcers, etc.), ischemic/vascular events (e.g., myocardial infarct, stroke, shock, hemorrhage, coagulopathy, etc.), infections (e.g., cellulitis, pneumonia, meningitis, SIRS, etc.), neoplasia (e.g., breast cancer, lung cancer, lymphoma, etc.), immunologic/autoimmune conditions (e.g., graft vs. host disease, multiple sclerosis, diabetes, inflammatory bowel disease, lupus erythematosus, rheumatoid arthritis, psoriasis, etc.), degenerative diseases (e.g., osteoporosis, osteoarthritis, Alzheimer's disease, etc.), congenital/genetic diseases (e.g., epidermolysis bullosa, osteogenesis imperfecta, muscular dystrophies, lysosomal storage diseases, Huntington's disease, etc.), adverse drug effects (e.g., drug-induced hepatitis, drug-induced cardiac injury, etc.), toxic injuries (e.g., radiation exposure(s), chemical exposure(s), alcoholic hepatitis, alcoholic pancreatitis, alcoholic cardiomyopathy, cocaine cardiomyopathy, etc.), metabolic derangements (e.g., uremic pericarditis, metabolic acidosis, etc.), iatrogenic conditions (e.g., radiation-induced tissue injury, surgery-related complications, etc.), and/or idiopathic processes (e.g., amyotrophic lateral sclerosis, Parsonnage-Turner Syndrome, etc.) or any combination thereof. In some aspects, a disease can comprise graft-vs-host diseases (GvHD), Epidermolysis Bullosa (EB), junctional EB (JEB), EB simplex (EBS), congenital ichthyosis, congenital dyskeratosis, Recessive Dystrophic form of EB (RDEB), macular degeneration, Alzheimer's disease, aging, Type II diabetes, heart disease, osteoporosis, chronic skin wounds, diabetes-associated ulcers/wounds, connective tissue diseases such as Ehlers-Danlos Syndrome (EDS) or Marfan syndrome, cancer, or any combination thereof. In some aspects, a disease can also comprise an injury. An injury can comprise a burn, a broken bone, a concussion, a contusion, a fractured bone, a ruptured tendon, a torn ligament, punctured, scarped and/or cut skin, or any other injury known in the art. In some aspects, a disease can be Ehlers-Danlos Syndrome.

As used herein, the term “treating” or “treat” describes the management and care of a patient for the purpose of combating a disease, condition, or disorder and includes the administration of a compound of the present disclosure, or a pharmaceutically acceptable salt, polymorph or solvate thereof, to alleviate the symptoms or complications of a disease, condition or disorder, or to eliminate the disease, condition or disorder. The term “treat” can also include treatment of a cell in vitro or an animal model.

As used herein, the term “preventing,” “prevent,” or “protecting against” describes reducing or eliminating the onset of the symptoms or complications of such disease, condition or disorder.

As used herein, the terms “ameliorate”, “ameliorating” and grammatical variations thereof mean to decrease the severity of the symptoms of a disease in a subject.

The terms “effective amount” and “therapeutically effective amount” of an agent or compound are used in the broadest sense to refer to a nontoxic but sufficient amount of an active agent or compound to provide the desired effect or benefit.

The term “benefit” is used in the broadest sense and refers to any desirable effect and specifically includes clinical benefit as defined herein. Clinical benefit can be measured by assessing various endpoints, e.g., inhibition, to some extent, of disease progression, including slowing down and complete arrest; reduction in the number of disease episodes and/or symptoms; reduction in lesion size; inhibition (i.e., reduction, slowing down or complete stopping) of disease cell infiltration into adjacent peripheral organs and/or tissues; inhibition (i.e. reduction, slowing down or complete stopping) of disease spread; decrease of auto-immune response, which may, but does not have to, result in the regression or ablation of the disease lesion; relief, to some extent, of one or more symptoms associated with the disorder; increase in the length of disease-free presentation following treatment, e.g., progression-free survival; increased overall survival; higher response rate; and/or decreased mortality at a given point of time following treatment.

In any method, composition or kit of the present disclosure, TERT can be human TERT (hTERT).

In any method, composition or kit of the present disclosure, TERC can be human TERC (hTERC).

As used herein, a “subject” includes a mammal. The mammal can be any mammal, e.g., a human, a primate, a mouse, a rat, a dog, a cat, a cow, a horse, a goat, a camel, a sheep, a pig or any other mammal. In some aspects, a mammal can be a human. The subject can be a male or a female.

EXAMPLES Example 1—Levels of hTERC Transcripts are Higher in Induced Pluripotent Stem Cells as Compared to Fibroblasts

The levels of human telomerase RNA component (hTERC) transcripts in fibroblasts (FBs), induced pluripotent stem cells (iPSCs) were measured using the NanoString nCounter Gene Expression Assay. An approximately 2.4-4.6-fold upregulation of hTERC in iPSCs as compared to the parental F50 (human dermal fibroblast derived from a 50 year old individual) and FN2 (neonatal fibroblasts) lines, as shown in FIG. 4.

Example 2—Contacting Somatic Cells with Compositions of the Present Disclosure Increases the Level Of hTERC in the Somatic Cells

In this example, various cell lines were transfected with various compositions of the present disclosure.

Human dermal fibroblasts derived from a 50 year old individual (F50), neonatal human epidermal keratinocytes (HEKn) and GFP-expressing human mesenchymal stem/stromal cells (hMSC-GFP) were subjected to one transfection with 500 ng modified mRNA (mod-mRNA) encoding dCas9-VPR and 500 ng hTERC guide RNA (gRNA) (either independently or as a mix of 4 guides at a 1:1:1:1 ratio) using Lipofectamine® RNAiMAX™ (RNAiMAX) (Thermo Fisher Scientific). Cells were collected 24 post-transfection. Real time quantitative PCR reactions for human TERC RNA expression were then carried out using the Bio-Rad CFX Connect System. Data was analyzed using the ΔΔCt method.

To test the activation of the endogenous hTERC transcript, F50 cells were transfected with either 1 separate individual gRNA (g, g2, g3, and g4) plus mod-mRNA encoding dCas9-VPR or a mix of all 4 guides at a 1:1:1:1 ratio (gmix) plus mod-mRNA encoding dCas9-VPR. Levels of hTERC transcript were quantified using quantitative reverse transcription PCR. As shown in FIG. 5, the results indicate that even as little as only 1 single guide is sufficient to activate endogenous hTERC (g2, g4) to levels comparable to those achieved by the mix of 4 gRNAs. Transfection with the mix of 4 gRNAs and mod-mRNA encoding dCas9-VPR displayed the greatest increase in expression of hTERC, as shown in FIG. 5.

To monitor endogenous hTERC activation by dCas9-VPR across other cell lines, HEKn and hMSC-GFP cell lines were transfected with a mix of all 4 gRNAs plus mod-mRNA encoding dCas9-VPR. The expression of hTERC was then measured using quantitative reverse transcription PCR. The results are shown in FIG. 6. As shown in FIG. 6, the level of hTERC activation observed in all tested cells lines was comparable to that observed in iPSCs.

Example 2 Methods

Cell lines: 50 year-old human dermal fibroblast (F50, at passage 5), and neonatal human epidermal keratinocytes (HEKn, at passage 7) lines were obtained from ATCC. Human mesenchymal stem/stromal cells with GFP fluorescence (hMSC-GFP, at passage 15) were obtained from Cyagen. The F50 line was cultured in fibroblast expansion medium (FEM) comprised of DMEM/F12 supplemented with 5% human serum, 1× MEM non-essential amino acids solution, 55 μM of 2-mercaptoethanol (β-ME), 1× GlutaMAX™ supplement, plus antibiotics (all from Thermo Fisher Scientific), with 50 ug/ml ascorbic acid, 1 ng/ml hydrocortisone (both from Sigma), 12 ng/ml basic FGF (Gibco) and 5 ng/ml human EGF (Invitrogen). HEKn cells were cultured in EpiLife medium supplemented with EDGS and antibiotics (all from ThermoFisher). hMSCs were cultured in mesenchymal stem cell growth medium (MSCGM) (prepared as a kit from Cyagen).

Transfections: All transfections of fibroblasts and keratinocytes were performed using Opti-MEM®I Reduced Serum Medium (Opti-MEM) (Thermo Fisher Scientific) as a complexation buffer, while transfections of human mesenchymal stem/stromal cells (hMSCs) was performed using Opti-MEM with the pH adjusted to 8.2 (Opti-MEM-pH 8.2) as described in Kogut et al. Nature Communications, 2018. One transfection with 500 ng mod-mRNA encoding dCas9-VPR and 500 ng hTERC guide RNA (gRNA) (either individual (g1, g2, g3, g4) or a mix of 4 guides (gmix) at a 1:1:1:1 ratio) or 500 ng modified mRNA encoding dCas9-VPR alone was performed using Lipofectamine® RNAiMAX™ (RNAiMAX) (Thermo Fisher Scientific). RNA and RNAiMAX were first diluted in either Opti-MEM for keratinocyte and fibroblast transfections, or Opti-MEM-pH 8.2 for hMSC transfections. For mod-mRNA and/or gRNA transfections, 100 ng/μl RNA was diluted 5×, and 5 μl of RNAiMAX per microgram of mod-mRNAs and/or gRNA was diluted 10× using either Opti-MEM (for keratinocytes and fibroblasts) or Opti-MEM-pH 8.2 (for hMSCs). After dilution, these components were combined together and incubated for 15 min at room temperature (RT). After incubation at RT, transfection mixtures of mod-RNA mix and/or gRNA and RNAiMAX were applied to the cell cultures, in their respective media supplemented with 200 ng/ml B18R (eBioscience).

PCR: F50, HEKn, and hMSC-GFP cells collected 24 hours post-transfection. RNA was extracted using the RNeasy Plus Minikit (Qiagen). cDNA was synthesized using the iScript™ cDNA Synthesis Kit (BioRad). Quantitative PCR (QPCR) reactions for human TERC RNA were performed using SsoAdvanced™ Universal SYBR® Green Supermix. Data was analyzed using the ΔΔCt method.

Summary of Example 2: transfecting somatic cells with compositions of the present disclosure, more specifically a mod-mRNA encoding dCas9-VPR in combination with a plurality of gRNAs comprising 1 or more different gRNA species, can increase the expression of hTERC in the transfected cells, including to levels that are comparable to induced pluripotent stem cells.

Example 3—Transfecting Mod-RNA Encoding dCas9-VPR Alone does not Induce hTERC Expression in Target Cells

In this example, various cell lines were transfected with various compositions of the present disclosure, specifically a composition comprising only a mod-RNA encoding dCas9-VPR.

50 year-old human dermal fibroblasts (F50), and GFP-expressing human mesenchymal stem/stromal cells (hMSC-GFP) were subjected to one transfection with 500 ng mod-mRNA encoding dCas9-VPR using Lipofectamine® RNAiMAX™ (RNAiMAX) (Thermo Fisher Scientific). Cells were collected 24 hours (F50, hMSC-GFP) post-transfection. Quantitative reverse transcription PCR reactions for human TERC RNA expression were carried out using the Bio-Rad CFX Connect System and data was analyzed using the ΔΔCt method. The results are shown in FIG. 7. As shown in FIG. 7, the expression levels of hTERC does not increase when only a mod-RNA encoding dCas9-VPR is transfected into a target cell in the absence of any guide RNA.

Example 3 Methods

Cell lines: 50 year-old human dermal fibroblast (F50, at passage 5) were obtained from ATCC. Human mesenchymal stem/stromal cells with GFP fluorescence (hMSC-GFP, at passage p11) were obtained from Cyagen. F50 line was cultured in fibroblast expansion medium (FEM) comprised of DMEM/F12 supplemented with 5% human serum, 1× MEM non-essential amino acids solution, 55 μM of 2-mercaptoethanol (β-ME), 1× GlutaMAX™ supplement, plus antibiotics (all from Thermo Fisher Scientific), with 50 ug/ml ascorbic acid, 1 ng/ml hydrocortisone (both from Sigma), 12 ng/ml basic FGF (Gibco) and 5 ng/ml human EGF (Invitrogen). hMSCs were cultured in mesenchymal stem cell growth medium (MSCGM) (prepared as a kit from Cyagen).

Transfections: All transfections of fibroblasts were performed using Opti-MEM as a complexation buffer, while transfections of human mesenchymal stem/stromal cells (hMSCs) was performed Opti-MEM-pH 8.2 One transfection with 500 ng mod-mRNA encoding dCas9-VPR alone was performed using Lipofectamine® RNAiMAX™ (RNAiMAX) (Thermo Fisher Scientific). RNA and RNAiMAX were first diluted in appropriate Opti-MEM. For mod-mRNA transfections, 100 ng/μl RNA was diluted 5×, and 5 μl of RNAiMAX per microgram of mod-mRNAs was diluted 10× using appropriate Opti-MEM. After dilution, these components were combined together and incubated for 15 min at RT. After incubation at RT, transfection mixtures of mod-RNA mix and RNAiMAX were applied to the cell cultures, in their respective media supplemented with 200 ng/ml B18R (eBioscience).

PCR: F50 and hMSC-GFP cells were collected 24 hours post-transfection. RNA was extracted using the RNeasy Plus Minikit (Qiagen). cDNA was synthesized using the iScript™ cDNA Synthesis Kit (BioRad). QPCR reactions for human TERC RNA were performed using SsoAdvanced™ Universal SYBR® Green Supermix. Data was analyzed using the Δ66 Ct method. P values were calculated using a paired, two-tailed Student's t-test. *P≤0.05, **P≤0.01, ***P≤0.001.

Example 3 summary: Increases in hTERC expression using the methods and the compositions of the present disclosure are dependent on specific targeting of a DNA-targeting molecule comprising a transactivation domain, for example through the co-administration of at least one guide RNA.

Example 4—The Methods and Compositions of the Present Disclosure Cause an Increase in Population Doubling (PD) of Senescent Fibroblast Cells

In this example, senescent fibroblast cells were contacted with compositions of the present disclosure using methods of the present disclosure.

50 year-old human dermal fibroblast (F50) line was obtained from ATCC, and subsequently cultured until 90% of cells displayed the senescent phenotype as previously described in Kogut et al, Nature Communications, 2018. Briefly, the senescent phenotype can include an enlargement of cellular morphology and upwards of about 90% positivity for senescence-associated β-galactosidase. The F50 line was thawed (F50S, at passage 15, 32.5 PD) and cultured in FEM: DMEM/F12 supplemented with 5% human serum, 1× MEM non-essential amino acids solution, 55 μM of 2-mercaptoethanol (β-ME), 1× GlutaMAX™ supplement, plus antibiotics (all from Thermo Fisher Scientific), with 50 ug/ml ascorbic acid, 1 ng/ml hydrocortisone (both from Sigma), 12 ng/ml basic FGF (Gibco) and 5 ng/ml human EGF (Invitrogen). Initially, 10 k fibroblasts (F50S p15, 32.5 PD) were seeded, per well.

FIG. 8 shows a schematic of the transfection regimen of fibroblasts using rejuvenating compositions of the present disclosure. Initially, 10 k senescent fibroblasts (F50S, p15, 32.5 PD) were seeded, per well. The cells were first pre-treated with three sequential transfections with 500 ng mod-mRNA encoding hTERT. After pretreatment, four sequential transfection series with 500 ng mod-mRNA encoding hTERT followed by 500 ng mod-mRNA encoding dCas9-VPR and 500 ng human TERC guide RNA (gRNA) (4 guides, 1:1:1:1 ratio) the next day were performed using Lipofectamine® RNAiMAX™ (RNAiMAX) (Thermo Fisher Scientific). RNA and RNAiMAX were first diluted in Opti-MEM®I Reduced Serum Medium (Opti-MEM). For mod-mRNA and/or gRNA transfections, 100 ng/μl RNA was diluted 5×, and 5 μl of RNAiMAX per microgram of mod-mRNAs and/or gRNA were diluted 10× using Opti-MEM. After dilution, these components were combined together and incubated for 15 min at RT. After incubation at RT, transfection mixtures of mod-RNA mix and/or gRNA and RNAiMAX were applied to the cell culture, in FEM supplemented with 200 ng/ml B18R (eBioscience). The medium was changed after overnight incubation after each transfection.

The cumulative population doubling of non-treated senescent fibroblasts (F50S, p15, 32.5 starting PD) and the same cells treated with the rejuvenating composition (using the regimen put forth in FIG. 8) was measured. When cells reached 70-80% confluence, they were typsinized, counted using a hemocytometer, and passaged. PD was calculated as the log of the ratio of the final count (N) to the starting (baseline) count (X₀) divided by the log of 2; that is: PD=[log(N ÷X₀)]÷log 2. P values were calculated using a paired, two-tailed Student's t-test. *P≤0.05, **P≤0.01, ***P≤0.001. The results are shown in FIG. 9. As shown in FIG. 9, the treatment with the rejuvenating composition of the present disclosure results in the increased population doubling.

Example 4 summary: The compositions and methods of the present disclosure can be used to rejuvenate senescent cells, including senescent fibroblasts, leading to an increase in the total number of population doublings exhibited by the treated cells.

Example 5—the Methods and Compositions of the Present Disclosure Increase Telomere Length and Mitochondrial DNA Amount in Transfected Target Cells

In this example, various cell lines (low passage and senescent 50 year-old human dermal fibroblasts (F50 and F50S respectively), human mesenchymal stem/stromal cells (hMSCs) and human keratinocytes) were transfected with various compositions of the present disclosure using various methods of the present disclosure. Changes in telomere length in each cell line were then measured.

Low passage and senescent 50 year-old human dermal fibroblasts: 50 year-old human dermal fibroblast (F50) lines were obtained from ATCC, and subsequently cultured until 90% of cells displayed the senescent phenotype as previously described in Kogut et al, Nature Communications, 2018. Breifly, the senescent phenotype can include an enlargement of cellular morphology and upwards of about 90% positivity for senescence-associated β-galactosidase. The F50 lined was thawed (F50S, at passage 15, 32.5 PD) and cultured in FEM: DMEM/F12 supplemented with 5% human serum, 1× MEM non-essential amino acids solution, 55 μM of 2-mercaptoethanol (β-ME), 1× GlutaMAX™ supplement, plus antibiotics (all from Thermo Fisher Scientific), with 50 ug/ml ascorbic acid, 1 ng/ml hydrocortisone (both from Sigma), 12 ng/ml basic FGF (Gibco) and 5 ng/ml human EGF (Invitrogen). Initially, 10 k fibroblasts (F50 p3-4 or F50S p15, 32.5 PD) were seeded, per well.

FIG. 8 shows a schematic of a transfection regimen of fibroblasts using a rejuvenating composition of the present disclosure. Initially, 10 k senescent fibroblasts (F50S, p15, 32.5 PD) were seeded, per well. The cells were first pre-treated with three sequential transfections with 500 ng mod-mRNA encoding hTERT. After pretreatment, four sequential transfection series with 500 ng mod-mRNA encoding hTERT followed by 500 ng mod-mRNA encoding dCas9-VPR and 500 ng human TERC guide RNA (gRNA) (4 guides, 1:1:1:1 ratio) the next day were performed using Lipofectamine® RNAiMAX™ (RNAiMAX) (Thermo Fisher Scientific). RNA and RNAiMAX were first diluted in Opti-MEM®I Reduced Serum Medium (Opti-MEM). For mod-mRNA and/or gRNA transfections, 100 ng/μl RNA was diluted 5×, and 5 μl of RNAiMAX per microgram of mod-mRNAs and/or gRNA were diluted 10× using Opti-MEM. After dilution, these components were combined together and incubated for 15 min at RT. After incubation at RT, transfection mixtures of mod-RNA mix and/or gRNA and RNAiMAX were applied to the cell culture, in FEM supplemented with 200 ng/ml B18R (eBioscience). The medium was changed after overnight incubation after each transfection.

FIG. 10 shows a schematic of an alternative transfection regimen of fibroblasts using a rejuvenating composition of the present disclosure. F50 fibroblasts were plated in FEM at 15K cells per well of a 6-well format dish. Four transfection series with 500 ng mod-mRNA encoding hTERT together with 200 ng mod-mRNA encoding dCas9-VPR and 500 ng hTERC guide RNA (gRNA) (1 selected guide) were performed every 4 days using Lipofectamine® RNAiMAX™ (RNAiMAX) (Thermo Fisher Scientific). RNA and RNAiMAX were first diluted in Opti-MEM®I Reduced Serum Medium (Opti-MEM). For mod-mRNA and/or gRNA transfections, 100 ng/μl RNA was diluted 5×, and 5 μl of RNAiMAX per microgram of mod-mRNAs and/or gRNA were diluted 10× using Opti-MEM. After dilution, these components were combined together and incubated for 15 min at RT. After incubation at RT, transfection mixtures of mod-RNA mix and/or gRNA and RNAiMAX were applied to the cell culture, in FEM supplemented with 200 ng/ml B18R (eBioscience). The medium was changed after overnight incubation after each transfection.

Human mesenchymal stem/stromal cells: Human mesenchymal stem/stromal cells (hMSCs) were cultured in mesenchymal stem cell growth medium (MSCGM) (prepared as a kit from Cyagen) under low O₂ (5%). All transfections of hMSCs were performed using Opti-MEM with the pH adjusted to 8.2 (Opti-MEM-pH 8.2) as described in Kogut et al. Nature Communications, 2018.

FIG. 11 shows a schematic of the transfection regimen of hMSCs using rejuvenating compositions of the present disclosure. A pre-treatment of 3 transfections with 500 ng mod-mRNA encoding human TERT was performed using Lipofectamine® RNAiMAX™ (RNAiMAX) (Thermo Fisher Scientific). Following pre-treatment with hTERT mod-mRNA transfections, four sequential transfection series with 500 ng mod-mRNA encoding hTERT followed by 500 ng mod-mRNA encoding dCas9-VPR and 500 ng human TERC guide RNA (gRNA) (4 guides, 1:1:1:1 ratio) the next day were performed using Lipofectamine® RNAiMAX™ (RNAiMAX) (Thermo Fisher Scientific). RNA and RNAiMAX were first diluted in Opti-MEM-pH 8.2. For mod-mRNA transfections, 100 ng/μl RNA was diluted 5×, and 5 μl of RNAiMAX per microgram of mod-mRNAs was diluted 10× using Opti-MEM-pH 8.2. After dilution, these components were combined together and incubated for 15 min at RT. After incubation at RT, transfection mixtures of mod-RNA mix and RNAiMAX were applied to the cell culture, in respective media supplemented with 200 ng/ml B18R. The medium was changed after overnight incubation after each transfection.

Human keratinocytes: Human neonatal epidermal keratinocytes (HEKn) were cultured in EpiLife medium supplemented with EDGS and antibiotics (all from ThermoFisher). All transfections of HEKs were performed using Opti-MEM with no pH adjustment.

FIG. 11 shows a schematic of the transfection regimen of HEKn using rejuvenating compositions of the present disclosure. A pre-treatment of 3 transfections with 100 ng mod-mRNA encoding human human TERT was performed using Lipofectamine® RNAiMAX™ (RNAiMAX) (Thermo Fisher Scientific). RNA and RNAiMAX were first diluted in Opti-MEM®I Reduced Serum Medium (Opti-MEM) (Thermo Fisher Scientific). For mod-mRNA transfections, 100 ng/μl RNA was diluted 5×, and 5 μl of RNAiMAX per microgram of mod-mRNAs was diluted 10× using Opti-MEM. After dilution, these components were combined together and incubated for 15 min at RT. After incubation at RT, transfection mixtures of mod-RNA mix and RNAiMAX were applied to the cell culture, in EpiLife medium supplemented with 200 ng/ml B18R (eBioscience). Following pre-treatment of the HEKn cell line, 4 sequential transfection series with 100 ng mod-mRNA encoding hTERT followed by 100 ng mod-mRNA encoding dCas9-VPR+100 ng gRNA mix the next day were performed using Lipofectamine®RNAiMAX™ (RNAiMAX) (Thermo Fisher Scientific). RNA and RNAiMAX were first diluted in Opti-MEM (Thermo Fisher Scientific). For mod-mRNA and/or gRNA transfections, 100 ng/μl RNA was diluted 5×, and 5 μl of RNAiMAX per microgram of mod-mRNAs and/or gRNA were diluted 10× using Opti-MEM. After dilution, these components were combined together and incubated for 15 min at RT. After incubation at RT, transfection mixtures of mod-RNA mix and/or gRNA and RNAiMAX were applied to the cell culture, in EpiLife supplemented with 200 ng/ml B18R (eBioscience). The medium was changed after overnight incubation after each transfection.

The F5OS cells were collected 3 days after the last transfection. Genomic DNA (gDNA) was extracted using the DNeasy® Blood and Tissue Kit (Qiagen). Quantitative PCR reactions for relative telomere length in treated and untreated cells were performed using SsoAdvanced™ Universal SYBR® Green Supermix. The results are shown in FIG. 12.

The F50 cells were collected 3 days after the last transfection. Genomic DNA (gDNA) was extracted using the Quick-DNA™ Miniprep Kit (Zymo Research). Quantitative PCR was used to determine changes in average telomere length in treated and untreated cells based on ScienCell's Absolute Human Telomere Length Quantification and Mitochondrial DNA Copy Number qPCR Assay Kit (#8958). The telomere primer set recognizes and amplifies telomere length by comparing samples to reference genomic DNA containing a 100 base pair (bp) telomere sequence located on human chromosome 17 (provided by kit). Primer-probe real-time PCR was performed using BioRad's CFX96 Real-Time System (BioRad, Hercules, Calif.). The results are shown in FIG. 13.

The hMSCs and HEKn cells were collected 3 days after the last transfection. Genomic DNA (gDNA) was extracted using the DNeasy® Blood and Tissue Kit (Qiagen). Quantitative PCR was used to determine changes in average telomere length in treated and untreated cells based on ScienCell's Absolute Human Telomere Length Quantification and Mitochondrial DNA Copy Number qPCR Assay Kit (#8958). The telomere primer set recognizes and amplifies telomere length by comparing samples to reference genomic DNA containing a 100 base pair (bp) telomere sequence located on human chromosome 17 (provided by kit). Primer-probe real-time PCR was performed using BioRad's CFX96 Real-Time System (BioRad, Hercules, Calif.). The results are shown in FIG. 14.

As shown in FIG. 12, FIG. 13 and FIG. 14, F50S, F50, hMSCs and HEKn cells treated with the rejuvenating compositions of the present disclosure displayed increased telomere length as compared to untreated control cells. Moreover, in the case of the treated F50S and HEKn cells, the telomere lengths exceeded the telomere lengths measured in F50-derived induced pluripotent stem cells (F50-iPSCs).

Quantitative PCR was used to determine changes in mitochondrial DNA copy number using ScienCell's Absolute Human Telomere Length Quantification and Mitochondrial DNA Copy Number Dual Quantification qPCR Assay Kit (#8958). The mtDNA primer set recognizes and amplifies one of the most conserved regions on human mtDNA and will not amplify any off-target sequence on nuclear genomic DNA. The single copy reference (SCR) primer set recognizes and amplifies a 100 bp-long region on human chromosome 17 and serves as reference for data normalization. Primer-probe real-time PCR was performed using BioRad's CFX96 Real-Time System (BioRad, Hercules, Calif.). The results are shown in FIG. 15. As shown in FIG. 15, F50S, hMSCs and HEKn cells treated with the rejuvenating compositions of the present disclosure displayed increased mitochondrial DNA copy number as compared to untreated cells.

Summary of Example 5: the compositions and methods of the present disclosure can be used to rejuvenate various cell types, including low passage and senescent fibroblasts, human mesenchymal stem/stromal cells and human epidermal keratinocytes, leading to an increase in telomere length and mitochondrial DNA amount in treated cells.

Example 6—the Methods and Compositions of the Present Disclosure Reactivate Telomerase Activity in Fibroblasts

In this example, 50 year-old human fibroblasts (F50) were transfected with various compositions of the present disclosure. The telomerase activity in the transfected target cells, as well as control cells, was analyzed.

Fifty year-old human dermal fibroblast (F50 passage 6) were cultured in fibroblast expansion medium (FEM) comprised of DMEM/F12 supplemented with 5% human serum, 1× MEM non-essential amino acids solution, 55 μM of 2-mercaptoethanol (β-ME), 1× GlutaMAX™ supplement, plus antibiotics (all from Thermo Fisher Scientific), with 50 ug/ml ascorbic acid, 1 ng/ml hydrocortisone (both from Sigma), 12 ng/ml basic FGF (Gibco) and 5 ng/ml human EGF (Invitrogen). As untreated control lines, the F50-iPSC line was cultured in mTeSR™1 Media supplemented with 1× mTeSR™1 supplement (StemCell Technologies) plus antibiotics (Thermo Fisher Scientific) on plates coated with Matrigel coating matrix (Corning).

Two sequential transfections with either 3 ug mod-mRNA encoding hTERT or 3 ug mod-mRNA encoding hTERT with 3 ug mod-mRNA encoding dCas9-VPR+500 ng gRNA mix were performed using Lipofectamine® RNAiMAX™ (RNAiMAX) (Thermo Fisher Scientific). RNA and RNAiMAX were first diluted in Opti-MEM®I Reduced Serum Medium (Opti-MEM) (Thermo Fisher Scientific). For mod-mRNA transfections, 100 ng/μl RNA was diluted 5×, and 5 μl of RNAiMAX per microgram of mod-mRNAs was diluted 10× using Opti-MEM. After dilution, these components were combined together and incubated for 15 min at RT. After incubation at RT, transfection mixtures of mod-RNA and/or gRNA and RNAiMAX were applied to the cell culture, in FEM supplemented with 200 ng/ml B18R (eBioscience). The medium was changed after overnight incubation after each transfection.

Telomerase activity was measured with the TRAPeze® Telomerase Detection Kit (Millipore) according to the manufacturer's instructions. CHAPS (1×) lysis buffer was used to obtain extracts from, positive control cells (kit provided), an iPSC line derived from F50 (F50-iPSC), fibroblasts (F50), and fibroblasts (F50) treated with two sequential transfections of 3 ug hTERT only or 3 ug hTERT with 3 ug dCas9-VPR+3 ug gRNA mix. About 10,000 cells were assayed for each telomeric repeat amplification protocol assay, and 1,500 cell equivalents were loaded into each well of a 15% non-denaturing TBE (Tris borate, EDTA)-Urea polyacrylamide gel. Each sample was heat inactivated for 10 min at 85° C. to assess the background of the assay.

The results of the telomerase activity assay are shown in FIG. 16. Brighter products are indicative of higher activity. As show in in FIG. 16, the combined treatment with mod-mRNA encoding hTERT, mod-mRNA encoding dCas9-VPR and hTERC-specific gRNAs resulted in a higher level of telomerase activity as compared to untreated iPSCs or F50 cells transfected with only mod-mRNA encoding hTERT treatment alone.

Example 6 summary: The compositions and methods of the present disclosure can reactivate and increase telomerase activity in target cells, thereby rejuvenating the target cells.

Example 7—the Compositions of The Present Disclosure Facilitate Single Cell Expansion

In this example, primary human adult fibroblasts were transfected with compositions of the present disclosure to determine if the methods and compositions of the present disclosure could support the expansion from a single cell.

Primary human adult fibroblasts were obtained from a skin biopsy. Adult fibroblasts were cultured in fibroblast expansion medium (FEM) comprised of DMEM/F12 supplemented with 5% human serum, 1× MEM non-essential amino acids solution, 55 μM of 2-mercaptoethanol (β-ME), 1× GlutaMAX™ supplement, plus antibiotics (all from Thermo Fisher Scientific), with 50 ug/ml ascorbic acid, 1 ng/ml hydrocortisone (both from Sigma), 12 ng/ml basic FGF (Gibco) and 5 ng/ml human EGF (Invitrogen). Individual patient-derived fibroblasts were plated and single cells selected using a (10×10 mm) PYREX® cloning cylinder.

FIG. 11 shows a schematic of the transfection regimen of the select, single fibroblasts using rejuvenating compositions of the present disclosure, with adjustments made for the reduced tissue culture surface area. The cells were first pre-treated with three sequential transfections with 50 ng mod-mRNA encoding hTERT. After pretreatment, four sequential transfection series with 50 ng mod-mRNA encoding hTERT followed by 50 ng mod-mRNA encoding dCas9-VPR and 50 ng hTERC guide RNA (gRNA) (4 guides, 1:1:1:1 ratio) the next day were performed using Lipofectamine® RNAiMAX™ (RNAiMAX) (Thermo Fisher Scientific). RNA and RNAiMAX were first diluted in Opti-MEM®I Reduced Serum Medium (Opti-MEM). For mod-mRNA and/or gRNA transfections, 100 ng/μl RNA was diluted 5×, and 5 μl of RNAiMAX per microgram of mod-mRNAs and/or gRNA were diluted 10× using Opti-MEM. After dilution, these components were combined together and incubated for 15 min at RT. After incubation at RT, transfection mixtures of mod-RNA mix and/or gRNA and RNAiMAX were applied to the cell culture, in FEM supplemented with 200 ng/ml B18R (eBioscience). The medium was changed after overnight incubation after each transfection.

Following the transfection regimen, each well was trypsinized and cells were transferred into one well of a 6-well tissue culture plate for further expansion in FEM. As shown in FIG. 17, one week after the last transfection, 2/10 wells of untreated cells expanded successfully and were able to be collected for gDNA extraction while 9/10 wells of treated fibroblasts expanded and collected.

Summary of Example 7: The compositions and methods of the present disclosure can facilitate the expansion of even single cells.

Example 8—the Compositions and Methods of the Present Disclosure Increase The Migration Activity of High Passage Human Mesenchymal Stem/Stromal Cells (hMSCs)

In this example, human mesenchymal stem/stromal cells (hMSCs) were transfected with compositions of the present disclosure to determine if the compositions and methods of the present disclosure can increase the migration activity of high passage hMSCs.

To measure migration activity of treated and untreated hMSCs, a Transendothelial Migration (TEM) assay was used. A schematic of the TEM assay is shown in FIG. 18. Briefly, Corning FluoroBlok cell culture inserts were pre-seeded with human endothelial cells (HUVEC). GFP+hMSCs are plated, and their migration through the HUVEC layer and pores of the FluoroBlok membrane was quantified over time via bottom-reading fluorescence microscopes such as the CellInsight CX7 High-Content Screening (HCS) Platform.

24-well Corning FluoroBlok™ Inserts were coated with collagen. After coating, human umbilical vein endothelial cells (HUVECs) were plated at 80K/cm² in ECM-2MV BulletKit™ media (Lonza) and allowed to attach in 5% CO2 incubation overnight. Following overnight incubation and successful attachment, media in the basal chamber was changed to Human Mesenchymal Stem Cell Growth Medium (Cyagen) supplemented with human recombinant EGF [10 ng/mL] (Stemcell Technologies). Human Mesenchymal Stem/Stromal Cells labeled with Green Fluorescent Protein (GFP) purchased from Cyagen were cultured for twelve passages (P12) and treated with the rejuvenating compositions of the present disclosure as described in FIG. 11 and Example 5, while control hMSCs were cultured without mRNA treatment. The rejuvenation procedure lasted for two passages bringing the passage number to P14. A portion of the rejuvenated hMSCs were frozen in a CoolCell LX™ overnight at −80° while the remaining cells were allowed to remain in culture. After two passages, the frozen cells were thawed and allowed to culture for further two passages.

The four conditions were as follows; Old high passage hMSCs (P20) were never rejuvenated or frozen; Young low passage hMSCs (P5) were a fresh vial of GFP labeled hMSCs (Cyagen) that was thawed allowed to attached overnight then lifted and run on the Transendothelial Migration Assay, frozen rejuvenated hMSCs (P17) that were frozen at P15 then thawed and allowed to culture for two passages and rejuvenated hMSCs that were never frozen but underwent five passages following the rejuvenation protocol. These four conditions were then added to the apical chamber of the FluoroBlok on top of the layer of attached HUVECs. Four fields of view from three replicates were obtained on ThermoScientific's CellInsight CX7 LED High-Content Screening (HCS) Platform. The CX7 HCS is designed to use brightfield, widefield and confocal microscopy for the entire fluorescence spectrum to rapidly capture and quantify high content data such as the kinetic analysis performed on Transendothelial Migration Assays (TEM). As shown in FIG. 19, the rejuvenated high passage hMSCs reached the saturation point significantly faster than untreated young hMSCs (40-50 ordinal time vs 130 ordinal time), while old high passage hMSCs (P20) showed poor migratory ability. A one-way ANOVA analysis exhibited significance of p<0.0001 between young and both groups rejuvenated hMSCs as compared to high passage hMSCs.

Example 8 summary: The compositions and methods of the present disclosure can rejuvenate hMSCs as evidenced by the increase in the migration activity of high passage hMSCs treated using the compositions and methods of the present disclosure.

Example 9—the Compositions and Methods of the Present Disclosure Restore the Level of Thiol Group Oxidation of Proteins in High Passage Senescent Human Mesenchymal Stem/Stromal Cells (hMSCs) to that Observed in Young Low Passage hMSCs

In this example, senescent high passage human mesenchymal stem/stromal cells (hMSCs) were transfected with compositions of the present disclosure to determine if the compositions and methods of the present disclosure can restore the level of thiol group oxidation of proteins in high passage senescent human mesenchymal stem/stromal cells (hMSCs) to that observed in young low passage hMSCs. Among amino acids, the sulphur-containing cysteine (Cys) is particularly prone to oxidation. This is due to the presence of the thiol moiety (—SH) in the side chain of Cys, which can easily form disulfide bonds with a different thiol moiety in response to oxidation. Reversible oxidation of Cys thiols regulate the activity of enzymes and ligand binding, as well as participate in redox signaling, which deregulation plays an essential role in the development of many human disease and aging.

Human mesenchymal stem/stromal cells (hMSCs) were cultured in mesenchymal stem cell growth medium (MSCGM) (prepared as a kit from Cyagen) under low O₂ (5%). The three conditions were as follows: Senescent high passage hMSCs (P14) were never rejuvenated; Young low passage hMSCs (P5) were a fresh vial of hMSCs that were thawed allowed to attached overnight then lifted and processed for the peptide analysis; Senescent high passage hMSCs treated at passage 12 with the rejuvenating compositions of the present disclosure. The rejuvenation procedure lasted for two passages bringing the passage number of treated senescent hMSCs to P14 before the peptide analysis was performed, matching untreated senescent hMSCs.

For hMSCS, a pre-treatment of 3 transfections with 500 ng mod-mRNA encoding human TERT was performed using Lipofectamine® RNAiMAX™ (RNAiMAX) (Thermo Fisher Scientific). Following pre-treatment with hTERT mod-mRNA transfections, three sequential transfection series with 500 ng mod-mRNA encoding hTERT followed by 500 ng mod-mRNA encoding dCas9-VPR and 500 ng hTERC guide RNA (gRNA) (1 selected guide) the next day were performed using Lipofectamine® RNAiMAX™ (RNAiMAX) (Thermo Fisher Scientific). RNA and RNAiMAX were first diluted in Opti-MEM-pH 8.2. For mod-mRNA transfections, 100 ng/μl RNA was diluted 5×, and 5 μl of RNAiMAX per microgram of mod-mRNAs was diluted 10× using Opti-MEM-pH 8.2. After dilution, these components were combined together and incubated for 15 min at RT. After incubation at RT, transfection mixtures of mod-RNA mix and RNAiMAX were applied to the cell culture, in respective media supplemented with 200 ng/ml B18R. The medium was changed after overnight incubation after each transfection.

Transfected senescent and un-transfected senescent and low passage young hMSCs were processed using iodoTMTsixplex Isobaric Mass Tag Labeling Kit (ThermoScientific). Resulted iodoTMT labeled peptide mix was analyzed by QExactive HF Orbitrap mass spectrometer with an Easy nLC 1000 UPLC system (Thermo Fischer Scientific). Peptide identifications were performed using MaxQuant program. Each MS/MS spectrum was analyzed against a human specific database (Uniprot). After this analysis, data files were exported and additionally analyzed with Perseus software for data of interest. Each experiment was repeated twice. MaxQuant and Perseus software were downloaded from Max Planck Institute of Biochemistry website.

The level of thiol group oxidation in senescent high passage hMSCs increased in 88 proteins and decreased in 31 proteins as compared to young hMSCs. The transfection of senescent hMSCs with rejuvenating compositions of the present disclosure resulted in the restoration of thiol group oxidation levels in approximately 90% of target proteins to the level observed in young cells. FIG. 20 shows representative results of the thiol group analysis in proteins whose thiol group oxidation levels increased (EIF2S1, TM9F3and USP14) and decreased (IGFB5) in senescent high passage hMSCs and the reversion of these thiol group oxidation levels to the young-like state in response to the treatment with the rejuvenating composition.

Example 9 summary: The compositions and methods of the present disclosure can rejuvenate hMSCs as evidenced by the restoration of the young-like level of protein thiol group oxidation in high passage hMSCs treated using the compositions and methods of the present disclosure.

Example 10—the Compositions and Methods of the Present Disclosure Reduce Senescence-Associated DNA Methylation in High Passage Senescent Human Mesenchymal Stem/Stromal Cells (hMSCs) and Human Neonatal Epidermal Keratinocytes (HEKn)

In this example, senescent high passage human mesenchymal stem/stromal cells (hMSCs) and senescent high passage human neonatal epidermal keratinocytes (HEKn) were transfected with compositions of the present disclosure to determine if the compositions and methods of the present disclosure can reduce the level of senescence-associated DNA methylation in these cells. Changes in DNA methylation have been recognized as one of the most common molecular alterations in aging and cellular senescence.

Human fibroblasts of different origin were cultured in FEM; human keratinocytes of different origin were cultured in EpiLife medium supplemented with EDGS and hMSCs were cultured in mesenchymal stem cell growth medium (MSCGM). The following cell types used for DNA methylation analysis were not treated with rejuvenating compositions: young low passage neonatal fibroblasts (P3), young low passage adult F50 fibroblasts (P3), young low passage neonatal keratinocytes HEKn (P3), young low passage fetal keratinocyte (P2), young low passage adult keratinocytes (P3), young umbilical cord-derived hMSCs (P2), senescent high passage F50S fibroblasts (P15), senescent high passage hMSCs (P13) and senescent high passage HEKn (P10). The treated group included senescent high passage HEKn and senescent high passage hMSCs treated with rejuvenating compositions as described in FIG. 11 and Example 5. After completing the treatment with compositions, the treated cells were expanded for additional 6 days. Genomic DNA (gDNA) was extracted from each cell culture using the Quick-DNA™ Miniprep Kit (Zymo Research) and subjected to DNA methylation analysis using the Illumina Infinium MathylationEPIC BeadChip Kit.

The DNA methylation data generated for cells of different types were analyzed using the R package “IlluminaHumanMethylationEPICanno.ilm10b2.hg19” and combined into three groups as follows: young cells, senescent (high passage) cells and senescent cells treated with rejuvenating compositions of the present disclosure. The young group included young low passage neonatal fibroblasts (P3), young low passage adult fibroblasts (P3), young low passage neonatal keratinocytes (P3), young low passage fetal keratinocyte (P2), young low passage adult keratinocytes (P3) and young umbilical cord-derived hMSCs (P2). The senescent group included senescent high passage F50S fibroblasts (P15), senescent high passage hMSCs (P13) and senescent high passage HEKn (P10). The treated group included senescent high passage HEKn and senescent high passage hMSCs treated with rejuvenating compositions. Cells of different type and origin were combined based on their senescence state to eliminate cell type-specific methylation differences among groups. The groups were compared using the 2-tailed t-test for two groups with unequal variance, and the degree of methylation was calculated as a fraction of methylated nucleotides at a site of interest, ranging from 0 to 1. Differential methylation sites were selected based on the largest difference in the degree of methylation for each group, and 9 DNA methylation sites were identified as the most methylated in senescent high passage cells irrespectively of the cell type of origin.

FIG. 21 depicts the location of 9 identified senescence-associated DNA marks and their associated genomic loci. All 9 sites showed an increase in DNA methylation levels in the senescence high passage group. The treatment of the cells from the senescent group with rejuvenating compositions of the present disclosure reduced the level of DNA methylation at all 9 sites to the level similar to that of the young cell group.

Example 10 summary: the compositions and methods of the present disclosure can be used to rejuvenate various cell types, including senescent human mesenchymal stem/stromal cells and human epidermal keratinocytes, leading to a reduction in senescence-association DNA methylation in treated cells. 

What is claimed is:
 1. A composition comprising: a) at least one first polynucleotide molecule comprising a nucleic acid sequence encoding at least a portion of telomerase reverse transcriptase (TERT); and b) at least one second polynucleotide molecule comprising a nucleic acid sequence encoding at least a portion of at least one DNA targeting polypeptide, wherein the DNA targeting polypeptide increases transcription of telomerase RNA component (TERC).
 2. A composition comprising: a) at least one first polynucleotide molecule comprising a nucleic acid sequence encoding at least a portion of telomerase reverse transcriptase (TERT); and b) at least one DNA targeting polypeptide, wherein the DNA targeting polypeptide increases transcription of telomerase RNA component (TERC).
 3. The composition of claim 1 or claim 2, wherein the at least one first polynucleotide molecule comprises an mRNA molecule encoding at least a portion of TERT.
 4. The composition of claim 1 or claim 2, wherein the at least one first polynucleotide molecule comprises a plasmid comprising a nucleic acid sequence encoding at least a portion of TERT operably linked to at least one promoter sufficient to drive expression of the at least one portion of TERT.
 5. The composition of claim 1, wherein the at least one second polynucleotide molecule comprises an mRNA molecule encoding at least a portion of at least one DNA targeting polypeptide.
 6. The composition of claim 1, wherein the at least one second polynucleotide molecule comprises a plasmid comprising a nucleic acid sequence encoding at least a portion of at least one DNA targeting polypeptide operably linked to at least one promoter sufficient to drive expression of the at least one portion of the at least one DNA targeting polypeptide.
 7. The composition of any of claims 1-6, wherein the DNA targeting polypeptide comprises at least one Cas9 molecule, at least one Cas9 variant molecule, at least one Cas9 ortholog molecule or any combination thereof.
 8. The composition of claim 7, wherein the Cas9 molecule, the Cas9 variant molecule or the Cas9 ortholog molecule is nuclease-deficient or nuclease-dead.
 9. The composition of claim 8, wherein the Cas9 variant molecule comprises eSpCas9 (K855A), eSpCas9 (1.0), eSpCas9 (1.1), SpCas9-HF1 (VP12), HypaCas9, xCas9, SpyFi Cas9, iSpy Cas9, iSpyMac, Cas9 (VQR), Cas9 (EQR), Cas9 (VRER), Cas9 (D1135E), Cas9(QQR1), SaCas9 (KKH), Nme1Cas9, Nme2Cas9, Nme3Cas9 or any combination thereof.
 10. The composition of claim 8, wherein the Cas9 ortholog molecule comprises Streptococcus pyogenes Cas9 (spCas9), Francisella novicida Cas9 (FnCas9), Staphylococcus aureus Cas9 (SaCas9), Neisseria meningitidis Cas9 (NmCas9; NmeCas9), Streptococcus thermophilus CRISPR1-Cas9 (St1Cas9), Streptococcus thermophilus CRISPR3-Cas9 (St3Cas9), Campylobacter jejuni Cas9 (CjCas9), Acidaminococcus sp. BV3L6 Cpf1 (AsCpf1), Lachnospiraceae bacterium ND2006 Cpf1 (LbCpf1), Streptococcus canis Cas9 (ScCas9), Treponema denticola Cas9 (TdCas9), Streptococcus macacae Cas9 (SmacCas9), Casϕ (Cas12j), Francisella tularensis subsp. novicida Cas9, Pasteurella multocida Cas9, Campylobacter lari CF89-12 Cas9, Mycoplasma gallisepticum str. F Cas9, Nitratifractor salsuginis str DSM 16511 Cas9, Parvibaculum lavamentivorans Cas9, Roseburia intestinalis Cas9, Neisseria cinerea Cas9, Gluconacetobacter diazotrophicus Cas9, Azospirillum B510 Cas9, Sphaerochaeta globus str. Buddy Cas9, Flavobacterium columnare Cas9, Fluviicola taffensis Cas9, Bacteroides coprophilus Cas9, Mycoplasma mobile Cas9, Lactobacillus farciminis Cas9, Streptococcus pasteurianus Cas9, Lactobacillus johnsonii Cas9, Staphylococcus pseudintermedius Cas9, Filifactor alocis Cas9, Legionella pneumophila str. Paris Cas9, Sutterella wadsworthensis Cas9, Corynebacter diphtheriae Cas9 or any combination thereof.
 11. The composition of claim 8, wherien the Cas9 ortholog molecule comprises a chimeric variant of Streptococcus pyogenes Cas9 (spCas9), Francisella novicida Cas9 (FnCas9), Staphylococcus aureus Cas9 (SaCas9), Neisseria meningitidis Cas9 (NmCas9; NmeCas9), Streptococcus thermophilus CRISPR1-Cas9 (St1Cas9), Streptococcus thermophilus CRISPR3-Cas9 (St3Cas9), Campylobacter jejuni Cas9 (CjCas9), Acidaminococcus sp. BV3L6 Cpf1 (AsCpf1), Lachnospiraceae bacterium ND2006 Cpf1 (LbCpf1), Streptococcus canis Cas9 (ScCas9), Treponema denticola Cas9 (TdCas9), Streptococcus macacae Cas9 (SmacCas9), Casϕ (Cas12j), Francisella tularensis subsp. novicida Cas9, Pasteurella multocida Cas9, Campylobacter lari CF89-12 Cas9, Mycoplasma gallisepticum str. F Cas9, Nitratifractor salsuginis str DSM 16511 Cas9, Parvibaculum lavamentivorans Cas9, Roseburia intestinalis Cas9, Neisseria cinerea Cas9, Gluconacetobacter diazotrophicus Cas9, Azospirillum B510 Cas9, Sphaerochaeta globus str. Buddy Cas9, Flavobacterium columnare Cas9, Fluviicola taffensis Cas9, Bacteroides coprophilus Cas9, Mycoplasma mobile Cas9, Lactobacillus farciminis Cas9, Streptococcus pasteurianus Cas9, Lactobacillus johnsonii Cas9, Staphylococcus pseudintermedius Cas9, Filifactor alocis Cas9, Legionella pneumophila str. Paris Cas9, Sutterella wadsworthensis Cas9, Corynebacter diphtheriae Cas9 or any combination thereof.
 12. The composition of any of claims 1-11, wherein the DNA targeting polypeptide comprises at least one TALE molecule, at least one zinc-finger molecule, at least one meganuclease molecule or any combination thereof
 13. The composition of any of claims 1-12, wherein the DNA targeting polypeptide comprises at least one transactivation molecule.
 14. The composition of claim 13, wherein the at least one transactivation molecule comprises at least one P65 molecule, at least one Rta molecule, at least one VP16 molecule, at least one VP64 molecule, at least one VP160 molecule, at least one VP64-P65-Rta (VPR) molecule, at least one SunTag peptide, at least one single guide RNA-MS2 (sgRNA-MS2) molecule or any combination thereof.
 15. The composition of any of claims 1-14, wherein the DNA targeting polypeptide comprises a DNA targeting ribonucleoprotein (RNP) complex.
 16. The composition of any of claims 1-15, wherein the DNA targeting polypeptide comprises at least one guide RNA.
 17. The composition of claim 13, wherein the transactivation molecule comprises at least one single guide RNA-MS2 (sgRNA-MS2) molecule.
 18. The composition of claim 17, wherein the at least one sgRNA-MS2 molecule comprises a nucleic acid sequence complementary to a nucleic acid sequence located upstream, within, or downstream of the endogenous TERC gene and at least about one, or at least about two, or at least about three, or at least about four, or at least about five, or at least about six, or at least about seven, or at least about eight, or at least about nine, or at least about ten MS2 RNA aptamers.
 19. The composition any of claims 1-18, wherein the DNA targeting polypeptide comprises a dCas9 molecule and a VPR molecule.
 20. The composition any of claims 1-19, wherein the DNA targeting polypeptide binds upstream of, 5′ to, within, downstream of or 3′ to the endogenous TERC gene.
 21. The composition any of claims 1-20, wherein the mRNA molecule is a modified mRNA molecule.
 22. The composition of claim 21, wherein the modified mRNA molecule comprises at least one modified ribonucleoside base.
 23. The composition of claim 22, wherein the modified ribonucleoside base comprises a pseudouridine (Ψ) residue, a 5-methylcytidine (m⁵C) residue or any combination thereof.
 24. The composition of claim 22, wherein the modified mRNA molecule comprises at least one modified nucleoside.
 25. The composition of claim 24, wherein the modified nucleoside comprise 5-methylcytidine (m⁵C), 5-methyluridine (m⁵U), N6-methyladenosine (m⁶A), inosine 2′-0-methylated nucleosides or any combination thereof.
 26. The composition of any of claims 1-25, further comprising a plurality of guide RNA (gRNA) molecules, wherein at least one gRNA in the plurality is complementary to a nucleic acid sequence located upstream, within, or downstream of the endogenous TERC gene.
 27. The composition of claim 26, wherein the plurality of gRNA molecules comprises at least about one, or at least about two, or at least about three, or at least about four, or at least about five, or at least about six, or at least about seven, or at least about eight, or at least about nine, or at least about ten distinct species of gRNA molecules, wherein each species has a different nucleic acid.
 28. The composition of any of claims 1-27, further comprising at least one plasmid comprising at least one nucleic acid sequence encoding at least one species of gRNA operably linked to at least one promoter sufficient to drive expression of the at least one species gRNA.
 29. The composition any of claims 26-28, wherein the plurality of gRNA molecules comprises a plurality of single guide RNA (sgRNA) molecules, crRNA:tracrRNA molecules, truncated sgRNA molecules, high fidelity scaffold gRNA molecules or any combination thereof.
 30. The composition of any of claims 26-29, wherein at least one guide RNA molecule is a modified guide RNA (mod gRNA) molecule.
 31. The composition of any of claims 26-30, wherein at least one guide RNA molecule comprises any sequence recited in Table 1 or Table
 2. 32. A composition comprising: a) at least one modified mRNA molecule comprising a nucleic acid sequence encoding at least a portion of human telomerase reverse transcriptase (hTERT); b) at least one modified mRNA molecule comprising a nucleic acid sequence encoding at least a portion of at least one DNA targeting polypeptide, wherein the at least one DNA targeting polypeptide comprises dCas9 and a VP64-P65-Rta (VPR) molecule; and c) a plurality of guide RNA (gRNA) molecules, wherein at least one gRNA in the plurality is complementary to a nucleic acid sequence located upstream of the endogenous hTERC gene.
 33. The composition of any of claims 1-32, further comprising at least one mRNA and/or polynucleotide encoding at least one rejuvenating factor.
 34. The composition of claim 33, wherein the rejuvenating factor comprises telomerase RNA component (TERC), telomerase associated reverse-transcriptase (TERT), protection of telomeres 1 (POT1), insulin-like growth factor 1 (IGF1), WD repeat containing antisense to TP53 (WRAP53), nuclear protein family A, member 3 (NOP3), heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1), shelterin complex subunit and telomerase recruitment factor (ACD/TPP1), TRF-1 interacting ankyrin-related ADP-ribose polymerase (TNKS), telomeric repeat binding factor 1 (TRF-1), telomeric repeat binding factor 2 (TRF-2), TERF1 interacting nuclear factor 2 (TIN2), telomeric repeat binding factor 2 (Rap1), Dyskerin Pseudouridine Synthase 1 (DKC1), ribonucleoprotein NHP2 or any combination thereof.
 35. A composition of any of claims 1-34, wherein TERT is human TERT (hTERT).
 36. A composition of any of claims 1-35, wherein TERC is human TERC (hTERC).
 37. A composition comprising at least one viral particle comprising the composition of any of claims 1-36.
 38. The composition of claim 37, wherein the at least one viral particle is an adeno-associated virus (AAV) particle, adenovirus particle, lentivirus particle, foamy-virus particle, herpes simplex virus (HSV) particle, retrovirus particle, alphavirus particle, flavivirus particle, rhabdovirus particle, measle virus particle, Newcastle disease virus particle, poxvirus particle, picornavirus particle, or any combination thereof.
 39. The composition of claim 38, wherein the at least one AAV particle is an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV2/1, AAV2/2, AAV2/3, AAV2/4, AAV2/5, AAV2/6, AAV2/7, AAV2/8, AAV2/9, AAV-DJ or AAV-DJ8 particle.
 40. The composition of claim 38, wherien the at least one retrovirus particle is an MMSV particle or an MSCV particle.
 41. The composition of claim 38, wherien the at least one lentivirus particle is an HIV-1 particle or an HIV-2 particle.
 42. The composition of claim 38, wherein the at least one alphavirus particle is an SFV particle, an SIN particle, a VEE particle, or an M1 particle.
 43. The composition of claim 38, wherien the at least one flavivirus particle is a Kunjin virus particle, a West Nile virus particle, or a Dengue virus particle particle.
 44. A composition comprising at least one exosome, microvesicle or liposome, wherein the at least one exosome, microvesicle or liposome comprises the composition of any of claims 1-34.
 45. A composition comprising least one nanoparticle, wherein the at least one nanoparticle comprises the composition of any of claims 1-36.
 46. The composition of claim 45, wherein the nanoparticle comprises a liposome, a micelle, a polymer-based nanoparticle, a lipid-polymer based nanoparticle, a metal based nanoparticle, a nanocrystal, a carbon nanotube based nanoparticle or a polymeric micelle.
 47. A kit comprising the composition of any of claims 1-45.
 48. A method of rejuvenating at least one cell, the method comprising contacting the at least one cell with the composition or kit of any of claims 1-47.
 49. The method of claim 48, further comprising expanding the at least one cell contacted with the composition or kit of any of claims 1-47 to produce a plurality of rejuvenated cells.
 50. A method of treating and/or preventing a disease in a subject comprising: a) contacting at least one cell with the composition or kit of any of claims 1-47; b) expanding the at least one cell contacted with the composition or kit of any of claims 1-47 to produce a plurality of rejuvenated cells; and c) administering the plurality of rejuvenated cells to the subject.
 51. A method of treating and/or preventing a disease in a subject comprising: a) contacting at least one cell with the composition or kit of any of claims 1-47; b) expanding the at least one cell contacted with the composition or kit of any of claims 1-47 to produce a plurality of rejuvenated cells; c) culturing the plurality of rejuvenated cells under conditions sufficient to transform the plurality of rejuvenated cells into at least one tissue or organ; and d) administering the at least one tissue or organ to the subject.
 52. A method of producing an in vitro tissue or organ comprising: a) contacting at least one cell with the composition or kit of any of claims 1-47; b) expanding the at least one cell contacted with the composition or kit of any of claims 1-47 to produce a plurality of rejuvenated cells; c) culturing the plurality of rejuvenated cells under conditions sufficient to transform the plurality of rejuvenated cells into at least one tissue or organ.
 53. A method of producing a plurality of rejuvenated edited cells comprising: a) contacting a plurality of cells with a gene editing system such that at least one gene in the genome of at least one cell in the plurality is edited, thereby producing at least one edited cell; b) isolating the at least one edited cell; c) contacting the isolated at least one edited cell with the composition or kit of any of claims 1-47; and d) expanding the at least one cell contacted with the composition or kit of any of claims 1-47 to produce a plurality of rejuvenated edited cell.
 54. A method of treating and/or preventing a disease in a subject comprising: a) contacting a plurality of cells with a gene editing system such that at least one gene in the genome of at least one cell in the plurality is edited, thereby producing at least one edited cell; b) isolating the at least one edited cell; c) contacting the isolated at least one edited cell with the composition or kit of any of claims 1-47; d) expanding the at least one cell contacted with the composition or kit of any of claims 1-47 to produce a plurality of rejuvenated edited cells; and e) administering to the subject the plurality of rejuvenated edited cells.
 55. A method of treating epidermolysis bullosa (EB) in a subject comprising: a) contacting a plurality of cells comprising keratinocytes, dermal fibroblasts, mesenchymal/stromal stem cells or any combination thereof with a gene editing system such that at least one gene in the genome of at least one cell in the plurality is edited, thereby producing at least one edited cell; b) isolating the at least one edited cell; c) contacting the isolated at least one edited cell with the composition or kit of any of claims 1-47; d) expanding the at least one cell contacted with the composition or kit of any of claims 1-47 to produce a plurality of rejuvenated edited cells; and e) administering to the subject the plurality of rejuvenated edited cells.
 56. The method of any of claims 48-55, wherein expanding the at least one cell comprises culturing the at least one cell using adjusted Opti-MEM, non-adjusted Opti-MEM, human serum, fetal bovine serum (FBS) or any combination thereof.
 57. The method of any of claim 48-56, wherein rejuvenating at least one cell comprises increasing the expression of TERC in the at least one cell, increasing the expression of TERT in the at least one cell, increasing the total number of population doublings exhibited by the at least one cell, increasing the length of telomeres in the at least one cell, increasing the mitochondrial DNA copy number in the at least one cell, increasing the amount of mitochondrial DNA in the at least one cell, increasing the number of mitochondria in the at least one cell, increasing the migration activity of the at least one cell, restoring the young-like state of thiol group oxidation levels in proteins in the at least one cell, reducing senescence-associated DNA methylation in the at least one cell or any combination thereof.
 58. The method of any of claim 48-57, wherein the at least one cell is a fibroblast, a keratinocyte, a mesenchymal stem/stromal cell, a peripheral blood mononuclear cell, a chimeric antigen receptor T cell (CAR-T cell), an endothelial cell, a chondrocyte, a muscle stem cell, a neural stem cell, a hepatocyte, a limbal stem cell, a retinal pigmented epithelial cell, a hematopoietic stem cell, a macrophage, a cardiomyocyte, a pancreatic cell, a β-cell or any combination thereof
 59. The method of any of claim 48-58, wherein the disease comprises graft-vs-host diseases (GvHD), autoimmune diseases, epidermolysis bullosa (EB), recessive dystrophic form of EB (RDEB), junctional EB (JEB), EB simplex (EBS), congenital ichthyosis, congenital dyskeratosis, macular degeneration, Parkinson's disease, Alzheimer's disease, aging, Type I and II diabetes, burns, chronic skin wounds, diabetes-associated ulcers/wounds, heart disease, osteoporosis, cancer, connective tissue diseases such as Ehlers-Danlos Syndrome (EDS) or Marfan syndrome, liver diseases, lung diseases, and any combination thereof.
 60. The method of any of claims 48-59, wherein contacting at least one cell comprises transfection, transduction, electroporation, nucleofection, at least one cell-penetrating peptide or any combination thereof.
 61. A method for rejuvenating at least one cell in a subject comprising administering to the subject at least one therapeutically effective amount of the composition of any of claims 1-46.
 62. A method for rejuvenating at least one subject comprising administering to the subject at least one therapeutically effective amount of the composition of any of claims 1-46.
 63. The method of any of claims 48-62, wherein the subject is a mammal.
 64. The method of claim 63, wherein the mammal is a human, a primate, a mouse, a rat, a dog, a cat, a cow, a horse, a goat, a camel, a sheep, a pig or any other mammal.
 65. The method of claim 64, wherein the mammal is a human. 